Methods and compositions for targeting the vasculature of solid tumors

ABSTRACT

The present invention relates generally to methods and compositions for targeting the vasculature of solid tumors using immunological- and growth factor-based reagents. In particular aspects, antibodies carrying diagnostic or therapeutic agents are targeted to the vasculature of solid tumor masses through recognition of tumor vasculature-associated antigens, such as, for example, through endoglin binding, or through the specific induction of endothelial cell surface antigens on vascular endothelial cells in solid tumors.

The U.S. government owns rights in the present invention pursuant to NIHGrant CA-28149 and NIH Grant CA54168.

The present application is a continuation-in-part of co-pending U.S.patent application Ser. No. 08/205,330, filed Mar. 2, 1994; which is acontinuation-in-part of U.S. patent application Ser. No. 07/846,349,filed Mar. 05, 1992, now abandoned. The entire text and figures of whichdisclosures are specifically incorporated by reference herein withoutdisclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and compositions fortargeting the vasculature of solid tumors using immunological and growthfactor-based reagents. In particular aspects, antibodies carryingdiagnostic or therapeutic agents are targeted to the vasculature ofsolid tumor masses through recognition of tumor vasculature-associatedantigens, such as endoglin, or through the specific induction of otherantigens on vascular endothelial cells in solid tumors.

2. Description of the Related Art

Over the past 30 years, fundamental advances in the chemotherapy ofneoplastic disease have been realized. While some progress has been madein the development of new chemotherapeutic agents, the more startlingachievements have been made in the development of effective regimens forconcurrent administration of drugs and our knowledge of the basicscience, e.g., the underlying neoplastic processes at the cellular andtissue level, and the mechanism of action of basic antineoplasticagents. As a result of the fundamental achievement, we can point tosignificant advances in the chemotherapy of a number of neoplasticdiseases, including choriocarcinoma, Wilm's tumor, acute leukemia,rhabdomyosarcoma, retinoblastoma, Hodgkin's disease and Burkitt'slymphoma, to name just a few. Despite the impressive advances that havebeen made in a few tumors, though, many of the most prevalent forms ofhuman cancer still resist effective chemotherapeutic intervention.

The most significant underlying problem that must be addressed in anytreatment regimen is the concept of "total cell kill." This conceptholds that in order to have an effective treatment regimen, whether itbe a surgical or chemotherapeutic approach or both, there must be atotal cell kill of all so-called "clonogenic" malignant cells, that is,cells that have the ability to grow uncontrolled and replace any tumormass that might be removed. Due to the ultimate need to developtherapeutic agents and regimens that will achieve a total cell kill,certain types of tumors have been more amenable than others to therapy.For example, the soft tissue tumors (e.g., lymphomas), and tumors of theblood and blood-forming organs (e.g., leukemias) have generally beenmore responsive to chemotherapeutic therapy than have solid tumors suchas carcinomas. One reason for this is the greater physical accessibilityof lymphoma and leukemic cells to chemotherapeutic intervention. Simplyput, it is much more difficult for most chemotherapeutic agents to reachall of the cells of a solid tumor mass than it is the soft tumors andblood-based tumors, and therefore much more difficult to achieve a totalcell kill. The toxicities associated with most conventional antitumoragents then become a limiting factor.

A key to the development of successful antitumor agents is the abilityto design agents that will selectively kill tumor cells, while exertingrelatively little, if any, untoward effects against normal tissues. Thisgoal has been elusive to achieve, though, in that there are fewqualitative differences between neoplastic and normal tissues. Becauseof this, much research over the years has focused on identifyingtumor-specific "marker antigens" that can serve as immunological targetsboth for chemotherapy and diagnosis. Many tumor-specific, orquasi-tumor-specific ("tumor-associated"), markers have been identifiedas tumor cell antigens that can be recognized by specific antibodies.Unfortunately, it is generally the case that tumor specific antibodieswill not in and of themselves exert sufficient antitumor effects to makethem useful in cancer therapy.

Over the past fifteen years, immunotoxins have shown great promise as ameans of selectively targeting cancer cells. Immunotoxins are conjugatesof a specific targeting agent typically a tumor-directed antibody orfragment, with a cytotoxic agent, such as a toxin moiety. The targetingagent directs the toxin to, and thereby selectively kills, cellscarrying the targeted antigen. Although early immunotoxins suffered froma variety of drawbacks, more recently, stable, long-lived immunotoxinshave been developed for the treatment of a variety of malignantdiseases. These "second generation" immunotoxins employ deglycosylatedricin A chain to prevent entrapment of the immunotoxin by the liver andhepatotoxicity (Blakey et al., 1987). They employ new crosslinkers whichendow the immunotoxins with high in vivo stability (Thorpe et al., 1988)and they employ antibodies which have been selected using a rapidindirect screening assay for their ability to form highly potentimmunotoxins (Till et al., 1988).

Immunotoxins have proven highly effective at treating lymphomas andleukemias in mice (Thorpe et al., 1988; Ghetie et al., 1991; Griffin etal., 1988) and in man (Vitetta et al., 1991). Lymphoid neoplasias areparticularly amenable to immunotoxin therapy because the tumor cells arerelatively accessible to blood-borne immunotoxins; also, it is possibleto target normal lymphoid antigens because the normal lymphocytes whichare killed along with the malignant cells during therapy are rapidlyregenerated from progenitors lacking the target antigens. In Phase Itrials where patients had large bulky tumor masses, greater than 50%tumor regressions were achieved in approximately 40% of the patients(Vitetta et al., 1991). It is predicted that the efficacy of theseimmunotoxins in patients with less bulky disease will be even better.

In contrast with their efficacy in lymphomas, immunotoxins have provedrelatively ineffective in the treatment of solid tumors such ascarcinomas (Weiner et al., 1989; Byers et al., 1989). The principalreason for this is that solid tumors are generally impermeable toantibody-sized molecules: specific uptake values of less than 0.001% ofthe injected dose/g of tumor are not uncommon in human studies (Sands etal., 1988; Epenetos et al., 1986). Furthermore, antibodies that enterthe tumor mass do not distribute evenly for several reasons. Firstly,the dense packing of tumor cells and fibrous tumor stromas present aformidable physical barrier to macromolecular transport and, combinedwith the absence of lymphatic drainage, create an elevated interstitialpressure in the tumor core which reduces extravasation and fluidconvection (Baxter et al., 1991; Jain, 1990). Secondly, the distributionof blood vessels in most tumors is disorganized and heterogeneous, sosome tumor cells are separated from extravasating antibody by largediffusion distances (Jain, 1990). Thirdly, all of the antibody enteringthe tumor may become adsorbed in perivascular regions by the first tumorcells encountered, leaving none to reach tumor cells at more distantsites (Baxter et al., 1991; Kennel et al., 1991). Finally,antigen-deficient mutants can escape being killed by the immunotoxin andregrow (Thorpe et al., 1988).

Thus, it is quite clear that a significant need exists for thedevelopment of novel strategies for the treatment of solid tumors. Oneapproach would be to target cytotoxic agents or coagulants to thevasculature of the tumor rather than to the tumor. Indeed, it has beenobserved that many existing therapies may already have, as part of theiraction, a vascular-mediated mechanism of action (Denekamp, 1990). Thepresent inventors propose that this approach offers several advantagesover direct targeting of tumor cells. Firstly, the target cells aredirectly accessible to intravenously administered therapeutic agents,permitting rapid localization of a high percentage of the injected dose(Kennel et al., 1991). Secondly, since each capillary provides oxygenand nutrients for thousands of cells in its surrounding `cord` of tumor,even limited damage to the tumor vasculature could produce an avalancheof tumor cell death (Denekamp, 1990; Denekamp, 1984). Finally, theoutgrowth of mutant endothelial cells lacking the target antigen isunlikely because they are normal cells.

For tumor vascular targeting to succeed, antibodies are required thatrecognize tumor endothelial cells but not those in normal tissues.Although several antibodies have been raised (Duijvestijn et al., 1987;Hagemeier et al., 1986; Bruland et al., 1986; Murray et al., 1989;Schlingemann et al., 1985) none has shown a high degree of specificity.

The antibodies termed TP-1 and TP-3, which were raised against humanosteosarcoma cells, have been reported to react with the same antigenpresent on proliferating osteoblasts in normal degenerating bone tissue.They also cross-react with capillary buds in a number of tumor types andin placenta, but apparently not with capillaries in any of the normaladult tissues examined (Bruland et al., 1986). It remains to be seenwhether the TP-1/TP-3 antigen is present on the surface of endothelialcells or whether the antibodies cross-react with gut endothelial cells,as was found with another antibody against proliferating endothelium(Hagemeier et al., 1986). This antibody described by Hagemeier andcolleagues (1986), termed EN7/44, reacts with a predominantlyintracellular antigen whose expression appears to be linked to migrationrather than proliferation (Hagemeier et al., 1986).

Immunotoxins in which the antibody portion is directed against thefibronectin receptor have also been proposed for use in killingproliferating vascular endothelial cells (Thorpe et al., 1990). However,intravenous administration of an immunotoxin containing dgA linked tothe anti-fibronectin receptor antibody termed PBl did not result inreduced vascularization of tumors (Thorpe et al., 1990). Unfortunately,further studies also revealed that fibronectin receptors were tooubiquitous to enable good targeting of tumor vasculature.

Other molecular markers have been described that are specific forendothelial cells, although not for tumor endothelial cells. Forexample, an endothelial-leukocyte adhesion molecule, termed ELAM-1, hasbeen identified that can be induced on the surface of endothelial cellsthrough the action of cytokines such as IL-1, TNF, lymphotoxin orbacterial endotoxin (Bevilacqua et al., 1987). However, the artcurrently lacks methods by which such inducible molecules could beeffectively employed in connection with an anti-cancer strategy. Thus,unfortunately, while vascular targeting presents promising theoreticaladvantages, no effective strategies incorporating these advantages havebeen developed.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the foregoing or otherdisadvantages in the prior art, by providing a series of novelapproaches for the treatment and/or diagnosis (imaging) of vascularizedsolid tumors. The invention rests in a general and overall sense on theuse of reagents, particularly immunological reagents, to targettherapeutic or diagnostic agents to tumor-associated vascularendothelial cells, alone or in combination with the direct targeting oftumor cells.

Such antibodies or growth factors will be referred to herein as"targeting agents". Thus, the targeting compounds of the invention maybe either targeting agent/therapeutic agent compounds or targetingagent/diagnostic agent compounds. Further, a targeting agent/therapeuticagent compound comprises a targeting agent operatively attached to atherapeutic agent, wherein the targeting agent recognizes and binds to atumor-associated endothelial cell marker. A targeting agent/diagnosticagent compound comprises a targeting agent operatively attached to adiagnostic agent, wherein the targeting agent recognizes and binds to atumor-associated endothelial cell marker. The targetingagent/therapeutic agent compounds of the invention may be produced usingeither standard recombinant DNA techniques or standard syntheticchemistry techniques, both of which are well known to those of skill inthe art.

In the case of diagnostic agents, the constructs will have the abilityto provide an image of the tumor vasculature, for example, throughmagnetic resonance imaging, x-ray imaging, computerized emissiontomography and the like.

In the case of therapeutic agents, constructs are designed to have acytotoxic or otherwise anticellular effect against the tumorvasculature, by suppressing the growth or cell division of the vascularendothelial cells. This attack is intended to lead to a tumor-localizedvascular collapse, depriving the tumor cells, particularly those tumorcells distal of the vasculature, of oxygen and nutrients, ultimatelyleading to cell death and tumor necrosis. In animal model systems, theinventors have achieved truly dramatic tumor regressions, with somecures being observed in combination therapy with anti-tumor directedtherapy.

It is proposed that the various methods and compositions of theinvention will be broadly applicable to the treatment or diagnosis ofany tumor mass having a vascular endothelial component. Typicalvascularized tumors are the solid tumors, particularly carcinomas, whichrequire a vascular component for the provision of oxygen and nutrients.Exemplary solid tumors to which the present invention is directedinclude but are not limited to carcinomas of the lung, breast, ovary,stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliarytract, colon, rectum, cervix, uterus, endometrium, kidney, bladder,prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cellcarcinomas, melanomas, gliomas, neuroblastomas, and the like.

A preferred method of the invention includes preparing an antibody thatrecognizes an antigen or other ligand associated with the vascularendothelial cells of the vascularized tumor mass, linking, oroperatively attaching the antibody to the selected agent to form anantibody-agent conjugate, and introducing the antibody-agent conjugateinto the bloodstream of an animal, such as a human cancer patient or atest animal in an animal model system. As used however, the term"antibody" is intended to refer broadly to any immunologic binding agentsuch as IgG, IgM, IgA, IgE, F(ab')₂, a univalent fragment such as Fab',Fab, Dab, as well as engineered antibodies such as recombinantantibodies, humanized antibodies, bispecific antibodies, and the like.

Alternatively, growth factors, rather than antibodies, may be utilizedas the reagents to target therapeutic or diagnostic agents totumor-associated vascular endothelial cells, alone, or in combinationwith the direct targeting of tumor cells. Any growth factor may be usedfor such a targeting purpose, so long as it binds to a tumor-associatedendothelial cell, generally by binding to a growth factor receptorpresent on the surface of such a tumor-associated endothelial cell.Suitable growth factors for targeting include, but are not limited to,VEGF/VPF (vascular endothelial growth factor/vascular permeabilityfactor), FGF (which, as used herein, refers to the fibroblast growthfactor family of proteins), TFGβ (transforming growth factor β), andpleitotropin. Preferably, the growth factor receptor to which thetargeting growth factor binds should be present at a higherconcentration on the surface of tumor-associated endothelial cells thanon non-tumor associated endothelial cells. Most preferably, the growthfactor receptor to which the targeting growth factor binds should,further, be present at a higher concentration on the surface oftumor-associated endothelial cells than on any non-tumor associated celltype.

The agent that is linked to the antibody or growth factor targetingagent will, of course, depend on the ultimate application of theinvention. Where the aim is to provide an image of the tumor, one willdesire to use a diagnostic agent that is detectable upon imaging, suchas a paramagnetic, radioactive or fluorogenic agent. Many diagnosticagents are known in the art to be useful for imaging purposes, as aremethods for their attachment to antibodies (see, e.g., U.S. Pat. Nos.5,021,236 and 4,472,509, both incorporated herein by reference). In thecase of paramagnetic ions, one might mention by way of example ions suchas chromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), with gadolinium being particularlypreferred. Ions useful in other contexts, such as X-ray imaging, includebut are not limited to lanthanum (III), gold (III), lead (II), andespecially bismuth (III). Moreover, in the case of radioactive isotopesfor therapeutic and/or diagnostic application, one might mentioniodine¹³¹, iodine¹²³, technicium^(99m), indium¹¹¹, rhenium¹⁸⁸,rhenium¹⁸⁶, gallium⁶⁷, copper⁶⁷, yttrium⁹⁰, iodine¹²⁵, or astatine²¹¹(for a review of the use of monoclonal antibodies in the diagnosis andtherapy of cancer, see Vaickus et al., 1991).

For certain applications, it is envisioned that the therapeutic agentswill be pharmacologic agents will serve as useful agents for attachmentto antibodies or growth factors, particularly cytotoxic or otherwiseanticellular agents having the ability to kill or suppress the growth orcell division of endothelial cells. In general, the inventioncontemplates the use of any pharmacologic agent that can be conjugatedto a targeting agent, preferably an antibody, and delivered in activeform to the targeted endothelium. Exemplary anticellular agents includechemotherapeutic agents, radioisotopes as well as cytotoxins. In thecase of chemotherapeutic agents, the inventors propose that agents suchas a hormone such as a steroid; an antimetabolite such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; ananthracycline; mitomycin C; a vinca alkaloid; demecolcine; etoposide;mithramycin; or an antitumor alkylating agent such as chlorambucil ormelphalan, will be particularly preferred. Other embodiments may includeagents such as a coagulant, a cytokine, growth factor, bacterialendotoxin or the lipid A moiety of bacterial endotoxin. In any event, itis proposed that agents such as these may, if desired, be successfullyconjugated to a targeting agent, preferably an antibody, in a mannerthat will allow their targeting, internalization, release orpresentation to blood components at the site of the targeted endothelialcells as required using known conjugation technology (see, e.g., Ghoseet al., 1983 and Ghose et al., 1987).

In certain preferred embodiments, therapeutic agents will includegenerally a plant-, fungus- or bacteria-derived toxin, such as an Achain toxins, a ribosome inactivating protein, α-sarcin, aspergillin,restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin,to mention just a few examples. The use of toxin-antibody constructs iswell known in the art of immunotoxins, as is their attachment toantibodies. Of these, a particularly preferred toxin for attachment toantibodies will be a deglycosylated ricin A chain. Deglycosylated ricinA chain is preferred because of its extreme potency, longer half-life,and because it is economically feasible to manufacture it a clinicalgrade and scale.

The present invention contemplates two separate and distinct approachesto the targeting of targeting agents, preferably antibodies, to thetumor vasculature. The first approach involves a targeting agent havinga binding affinity for a marker found, expressed, accessible to binding,or otherwise localized on the cell surfaces of tumor-associated vascularendothelial cells as compared to normal, non-tumor associatedvasculature. Further, certain markers for which a targeting agent has abinding affinity may be associated with the tumor-associated vasculaturerather than on the tumor-associated endothelial cells, themselves. Forexample, such markers may be located on basement membranes ortumor-associated connective tissue. It is preferred that targetingagents specific for such non-endothelial cell markers be operativelyattached to agents such as radioisotopes.

In the case of antibody targeting agents, such an approach involves thepreparation of an antibody having a binding affinity for antigenicmarkers found, expressed, accessible to binding or otherwise localizedon the cell surfaces of tumor-associated vascular endothelium ascompared to normal vasculature. Such a targeting agent, preferably anantibody, is then employed to deliver the selected diagnostic ortherapeutic agent to the tumor vasculature.

Naturally, where a therapeutic as opposed to diagnostic application isenvisioned it will be desirable to prepare and employ an antibody havinga relatively high degree of tumor vasculature selectivity, which mightbe expressed as having little or no reactivity with the cell surface ofnormal endothelial cells as assessed by immunostaining of tissuesections. Of course, with certain agents such as DNA synthesisinhibitors, and, more preferably, antimetabolites, the requirement forselectivity is not as necessary as it would be, for example, with atoxin, because a DNA synthesis inhibitor would have relatively littleeffect on the vascularation of normal tissues because the capillaryendothelial cells are not dividing. Further, such a degree ofselectivity is not a requirement for imaging purposes since cell death,and hence toxicity, is not the ultimate goal. In the case of diagnosticapplication, it is proposed that targeting agents, such as antibodies,having a reactivity for the tumor vasculature of at least two-foldhigher than for normal endothelial cells, as assessed by immunostaining,will be useful.

This aspect of the invention rests on the proposition that because oftheir proximity to the tumor itself, tumor-associated vascularendothelial cells are constantly exposed to many tumor-derived productssuch as cytokines (including lymphokines, monokines, colony-stimulatingfactors and growth factors), angiogenic factors, and the like, that willbind to and serve to selectively elicit the expression of tumorendothelium-specific cell surface markers. For use in the presentinvention, the anti-tumor vasculature antibodies may be directed to anyof the tumor-derived antigens which bind to the surface of vascularendothelial cells, and particularly to tumor-derived ligands, such asgrowth factors, which bind to specific cell surface receptors of theendothelial cells.

In connection with certain aspects of the invention, antibodies directedagainst tumor vasculature may be prepared by using endothelial cellsisolated from a tumor of an animal, or by "mimicking" the tumorvasculature phenomenon in vitro. As such, endothelial cells may besubjected to tumor-derived products, such as might be obtained fromtumor-conditioned media. Thus, this method involves generallystimulating endothelial cells with tumor-conditioned medium andemploying the stimulated endothelial cells as immunogens to prepare acollection of antibodies, for example, by utilizing conventionalhybridoma technology or other techniques, such as combinatorialimmunoglobulin phagemid libraries prepared from RNA isolated from thespleen of the immunized animal. One will then select from the antibodycollection an antibody that recognizes the tumor-stimulated vascularendothelium to a greater degree than it recognizes non-tumor-stimulatedvascular endothelium, and reacts more strongly with tumor-associatedendothelial cells in tissue sections than with those in normal adulthuman tissues, and producing the antibody, e.g., by culturing ahybridoma to provide the antibody.

Stimulated endothelial cells contemplated to be of use in this regardinclude, for example, human umbilical vein endothelial cells (HUVE),human dermal microvascular endothelial cells (HDEMC), human saphenousvein endothelial cells, human omental fat endothelial cells, other humanmicrovascular endothelial cells, human brain capillary endothelialcells, and the like. It is also contemplated that even endothelial cellsfrom another species may stimulated by tumor-conditioned media andemployed as immunogens to generate hybridomas to produce an antibodiesin accordance herewith, i.e., to produce antibodies which crossreactwith tumor-stimulated human vascular endothelial cells, and/orantibodies for use in pre-clinical models.

As used herein, "tumor-conditioned medium" is defined as a compositionor medium, such as a culture medium, which contains one or moretumor-derived cytokines, lymphokines or other effector molecules. Mosttypically, tumor-conditioned medium is prepared from a culture medium inwhich selected tumor cells have been grown, and will therefore beenriched in such tumor-derived products. The type of medium is notbelieved to be particularly important, so long as it at least initiallycontains appropriate nutrients and conditions to support tumor cellgrowth. It is also, of course, possible to extract and even separatematerials from tumor-conditioned media and employ one or more of theextracted products for application to the endothelial cells.

As for the type of tumor used for the preparation of the media, onewill, of course, prefer to employ tumors that mimic or resemble thetumor that will ultimately be subject to analysis or treatment using thepresent invention. Thus, for example, where one envisions thedevelopment of a protocol for the treatment of breast cancer, one willdesire to employ breast cancer cells such as ZR-75-1, T47D, SKBR3,MDA-MB-231. In the case of colorectal tumors, one may mention by way ofexample the HT29 carcinoma, as well as DLD-1, HCT116 or even SW48 orSW122. In the case of lung tumors, one may mention by way of exampleNCI-H69, SW2, NCI H23, NCI H460, NCI H69, or NCI H82. In the case ofmelanoma, good examples are DX.3, A375, SKMEL-23, HMB-2, MJM, T8 orindeed VUP. In any of the above cases, it is further believed that onemay even employ cells produced from the tumor that is to be treated,i.e., cells obtained from a biopsy.

Once prepared, the tumor-conditioned media is then employed to stimulatethe appearance of tumor endothelium-specific marker(s) on the cellsurfaces of endothelial cells, e.g., by culturing selected endothelialcells in the presence of the tumor-conditioned media (or productsderived therefrom). Again, it is proposed that the type of endothelialcell that is employed is not of critical importance, so long as it isgenerally representative of the endothelium associated with thevasculature of the particular tumor that is ultimately to be treated ordiagnosed. The inventors prefer to employ human umbilical veinendothelial cells (HUVE), or human dermal microvascular endothelialcells (HDMEC, Karasek, 1989), in that these cells are of human origin,respond to cytokine growth factors and angiogenic factors and arereadily obtainable. However, it is proposed that any endothelial cellthat is capable of being cultured in vitro may be employed in thepractice of the invention and nevertheless achieve benefits inaccordance with the invention. One may mention by way of example, cellssuch as EA.hy9.26, ECV304, human saphenous vein endothelial cells, andthe like.

Once stimulated using the tumor-derived products, the endothelial cellsare then employed as immunogens in the preparation of monoclonalantibodies. The technique for preparing monoclonal antibodies againstantigenic cell surface markers is quite straightforward, and may bereadily carried out using techniques well known to those of skill in theart, as exemplified by the technique of Kohler & Milstein (1975).Generally speaking, the preparation of monoclonal antibodies usingstimulated endothelial cells involves the following procedures. Cells orcell lines derived from human tumors are grown in tissue culture for ≧4days. The tissue culture supernatant (`tumor-conditioned medium`) isremoved from the tumor cell cultures and added to cultures of HUVEC at afinal concentration of 50% (v/v). After 2 days culture the HUVEC areharvested non-enzymatically and 1-2×10⁶ cells injected intraperitoneallyinto mice. This process is repeated three times at two-weekly intervals,the final immunization being by the intravenous route. Three days laterthe spleen cells are harvested and fused with SP2/0 myeloma cells bystandard protocols (Kohler & Milstein, 1975): Hybridomas producingantibodies with the appropriate reactivity are cloned by limitingdilution.

From the resultant collection of hybridomas, one will then desire toselect one of more hybridomas that produce an antibody that recognizesthe activated vascular endothelium to a greater extent than itrecognizes non-activated vascular endothelium. Of course, the ultimategoal is the identification of antibodies having virtually no bindingaffinity for normal endothelium. However, for imaging purposes thisproperty is not so critical. In any event, one will generally identifysuitable antibody-producing hybridomas by screening using, e.g., anELISA, RIA, IRMA, IIF, or similar immunoassay, against one or more typesof tumor-activated endothelial cells. Once candidates have beenidentified, one will desire to test for the absence of reactivity fornon-activated or "normal" endothelium or other normal tissue or celltype. In this manner, hybridomas producing antibodies having anundesirably high level of normal cross-reactivity for the particularapplication envisioned may be excluded.

The inventors have applied the foregoing technique successfully, in thatantibodies having relative specificity for tumor vascular endotheliumhave been prepared and isolated. In one particular example, theinventors employed the HT29 carcinoma to prepare the conditioned medium,which was then employed to stimulate HUVE cells in culture. Theresultant HT29-activated HUVE cells were then employed as immunogens inthe preparation of a hybridoma bank, which was ELISA-screened usingHT29-activated HUVE cells and by immunohistologic analysis of sectionsof human tumors and normal tissues. From this bank, the inventors haveselected antibodies that recognized a tumor vascular endothelial cellantigen.

The two most preferred monoclonal antibodies prepared by the inventorsusing this technique are referred to as tumor endothelial cell antibody4 and 11 (TEC4 and TEC11). The antigen recognized by TEC4 and TEC11 wasinitially believed to migrate as a doublet of about 43 kilodaltons (kD),as assessed by SDS/PAGE. However, as detailed herein, the presentinventors subsequently determined this antigen to be the moleculeendoglin, which migrates as a 95 kD species on SDS/PAGE under reducingconditions. The epitopes on endoglin recognized by TEC4 and TEC11 arepresent on the cell surface of stimulated HUVE cells, and only minimallypresent (or immunologically accessible) on the surface of non-stimulatedcells.

Monoclonal antibodies have previously been raised against endoglin(Gougos and Letarte, 1988; Gougos et al., 1992; O'Connel et al., 1992;Buhring et al., 1991). However, analyzing the reactivity with HUVEC orTCM-activated HUVEC cell surface determinants by FACS or indirectimmunofluorescence shows the epitopes recognized by TEC-4 and TEC-11 tobe distinct from those of a previous antibody termed 44G4 (Gougos andLetarte, 1988).

The TEC-4 and TEC-11 mAbs are envisioned to be particularly suitable fortargeting human tumor vasculature as they label capillary and venularendothelial cells moderately to strongly in a broad range of solidtumors (and in several chronic inflammatory conditions and fetalplacenta), but display relatively weak staining of vessels in themajority of normal, healthy adult tissues. TEC-11 is particularlypreferred as it shows virtually no reactivity with non-endothelialcells. Furthermore, both TEC-4 and TEC-11 are complement-fixing, whichimparts to them the potential to also induce selective lysis ofendothelial cells in the tumor vascular bed.

In addition to their use in therapeutic embodiments, TEC-4 and TEC-11antibodies may also be used for diagnostic, prognostic and imagingpurposes. For example, TEC-4 and TEC-11 may be employed to identifytumors with high vessel density, which is known to correlate withmetastatic risk and poor prognosis. This is a marked advance over thelaborious enumeration of capillaries labelled with pan-endothelial cellmarkers or the use of complex and subjective in vivo assays ofangiogenesis. Indeed, studies are disclosed herein which indicate thatTEC-4 and TEC-11 can distinguish between intraductal carcinoma in situ(CIS), an aggressive preneoplastic lesion and lobular CIS, which isassociated with a more indolent clinical course.

TEC-4 or TEC-11 antibodies may be linked to a paramagnetic, radioactiveor fluorogenic ion and employed in tumor imaging in cancer patients,where it is contemplated that they will result in rapid imaging due tothe location of endoglin on the luminal face of endothelial cells.Furthermore, TEC-4 and TEC-11 are of the IgM isotype, which limitsextravasation and enables more specific imaging of antigens in theintravascular compartment. This is in contrast to 44G4 which is an IgG1antibody.

The present invention therefore encompasses anti-endoglin antibodies andantibody-based compositions, including antibody conjugates linked toparamagnetic, radioactive or fluorogenic ions and anti-cellular agentssuch as anti-metabolites, toxins and the like, wherein the antibodiesbind to endoglin at the same epitope as either of the MAbs TEC-4 andTEC-11. Such antibodies may be of the polyclonal or monoclonal type,with monoclonals being generally preferred, especially for use inpreparing endoglin-directed antibody conjugates, immunotoxins andcompositions thereof.

The identification of an antibody or antibodies that bind to endoglin atthe same epitopes as TEC-4 or TEC-11 is a fairly straightforward matter.This can be readily determined using any one of variety of immunologicalscreening assays in which antibody competition can be assessed. Forexample, where the test antibodies to be examined are obtained from adifferent source to that of TEC-4 or TEC-11, e.g., a rabbit, or are evenof a different isotype, for example, IgG1 or IgG3, a competition ELISAmay be employed. In one such embodiment of a competition ELISA one wouldpre-mix TEC-4 or TEC-11 with varying amounts of the test antibodiesprior to applying to the antigen-coated wells in the ELISA plate. Byusing either anti-murine or anti-IgM secondary antibodies one will beable to detect only the bound TEC-4 or TEC-11 antibodies--the binding ofwhich will be reduced by the presence of a test antibody whichrecognizes the same epitope as either TEC-4 or TEC-11.

To conduct an antibody competition study between TEC-4 or TEC-11 and anytest antibody, one may first label TEC-4 or TEC-11 with a detectablelabel, such as, e.g., biotin or an enzymatic or radioactive label, toenable subsequent identification. In these cases, one would incubate thelabelled antibodies with the test antibodies to be examined at variousratios (e.g., 1:1, 1:10 and 1:100) and, after a suitable period of time,one would then assay the reactivity of the labelled TEC-4 or TEC-11antibodies and compare this with a control value in which no potentiallycompeting antibody (test) was included in the incubation.

The assay may be any one of a range of immunological assays based uponantibody binding and the TEC-4 or TEC-11 antibodies would be detected bymeans of detecting their label, e.g., using streptavidin in the case ofbiotinylated antibodies or by using a chromogenic substrate inconnection with an enzymatic label or by simply detecting theradiolabel. An antibody that binds to the same epitope as TEC-4 orTEC-11 will be able to effectively compete for binding and thus willsignificantly reduce TEC-4 or TEC-11 binding, as evidenced by areduction in labelled antibody binding. In the present case, aftermixing the labelled TEC-4 or TEC-11 antibodies with the test antibodies,suitable assays to determine the remaining reactivity include, e.g.,ELISAs, RIAs or western blots using human endoglin; immunoprecipitationof endoglin; ELISAs, RIAs or immunofluorescent staining of recombinantcells expressing human endoglin; indirect immunofluorescent staining oftumor vasculature endothelial cells; reactivity with HUVEC orTCM-activated HUVEC cell surface determinants indirectimmunofluorescence and FACS analysis. This latter method is mostpreferred and was employed to show that the epitopes recognized by TEC-4and TEC-11 are distinct from that of 44G4 (Gougos and Letarte, 1988).

The reactivity of the labelled TEC-4 or TEC-11 antibodies in the absenceof any test antibody is the control high value. The control low value isobtained by incubating the labelled antibodies with unlabelledantibodies of the same type, when competition would occur and reducebinding of the labelled antibodies. A significant reduction in labelledantibody reactivity in the presence of a test antibody is indicative ofa test antibody that recognizes the same epitope, i.e., one that"cross-reacts" with the labelled antibody. A "significant reduction" inthis aspect of the present application may be defined as a reproducible(i.e., consistently observed) reduction in binding of at least about100%-500 at a ratio of about 1:1, or more preferably, of equal to orgreater than about 90% at a ratio of about 1:100.

The present invention further encompasses antibodies which are specificfor epitopes present only on growth factor/growth factor receptorcomplexes, while being absent from either the individual growth factoror growth factor receptor. Thus, such antibodies will recognize and binda growth factor/growth factor receptor complex while not recognizing orbinding either the growth factor molecule or growth factor receptormolecule while these molecules are not in growth factor/growth factorreceptor complex form. A "growth factor/growth factor receptor complex"as used herein refers to a growth factor ligand bound specifically toits growth factor receptor, such as, by way of example only, a VEGF/VEGFreceptor complex.

As it is envisioned that the growth factor/growth factor receptorcomplexes to which these antibodies specifically bind are present insignificantly higher number on tumor-associated endothelial cells thanon non-tumor associated endothelial cells, such antibodies, when used astargeting agents, serve to further increase the targeting specificity ofthe agents of the invention. Such antibodies may be of the polyclonal ormonoclonal type, with monoclonals being generally preferred.

The second overall general approach presented by the present inventioninvolves the selective elicitation of vascular endothelial antigentargets on the surface of tumor-associated vasculature. This approachtargets known endothelial antigens that are present, or inducible, onthe cell surface of endothelial cells. The key to this aspect of theinvention is the successful manipulation of antigenic expression orsurface presentation such that the target antigen is expressed orotherwise available on the surface of tumor associated vasculature andnot expressed or otherwise available for binding, or at least to alesser extent, on the surface of normal endothelium.

A variety of endothelial cell markers are known that can be employed asinducible targets for the practice of this aspect of the invention,including endothelial-leukocyte adhesion molecule (ELAM-1; Bevilacqua etal., 1987); vascular cell adhesion molecule-1 (VCAM-1; Dustin et al.,1986); intercellular adhesion molecule-1 (ICAM-1; Osborn et al., 1989);the agent for leukocyte adhesion molecule-1 (LAM-1 agent), or even amajor histocompatibility complex (MHC) Class II antigen, such as HLA-DR,HLA-DP or HLA-DQ (Collins et al., 1984). Of these, the targeting ofELAM-1 or an MHC Class II antigen will likely be preferred fortherapeutic application, with ELAM-1 being particularly preferred, sincethe expression of these antigens will likely be the most direct topromote selectively in tumor-associated endothelium.

The targeting of an antigen such as ELAM-1 is the most straightforwardsince ELAM-1 is not expressed on the surfaces of normal endothelium.ELAM-1 is an adhesion molecule that can be induced on the surface ofendothelial cells through the action of cytokines such as IL-1, TNF,lymphotoxin or bacterial endotoxin (Bevilacqua et al., 1987). In thepractice of the present invention, the expression of ELAM-1 isselectively induced in tumor endothelium through the use of a bispecificantibody having the ability to cause the selective release of one ormore of the foregoing or other appropriate cytokines in the tumorenvironment, but not elsewhere in the body. This bispecific antibody isdesigned to cross-link cytokine effector cells, such as cells ofmonocyte/macrophage lineage, T cells and/or NK cells or mast cells, withtumor cells of the targeted solid tumor mass. This cross-linking isintended to effect a release of cytokine that is localized to the siteof cross-linking, i.e., the tumor.

Bispecific antibodies useful in the practice of this aspect of theinvention, therefore, will have a dual specificity, recognizing aselected tumor cell surface antigen on the one hand, and, on the otherhand, recognizing a selected "cytokine activating" antigen on thesurface of a selected leukocyte cell type. As used herein, the term"cytokine activating" antigen is intended to refer to any one of thevarious known molecules on the surfaces of leukocytes that, when boundby an effector molecule such as an antibody or a fragment thereof or anaturally-occurring agent or synthetic analog thereof, be it a solublefactor or membrane-bound counter-receptor on another cell, will promotethe release of a cytokine by the leukocyte cell. Examples of cytokineactivating molecules include CD14 and FcR for IgE, which will activatethe release of IL-1 and TNFα; and CD16, CD2 or CD3 or CD28, which willactivate the release of IFNγ and TNFβ, respectively.

Once introduced into the bloodstream of an animal bearing a tumor, sucha bispecific construct will bind to tumor cells within the tumor,cross-link those tumor cells with, e.g., monocytes/ macrophages thathave infiltrated the tumor, and thereafter effect the selective releaseof cytokine within the tumor. Importantly, however, withoutcross-linking of the tumor and leukocyte, the bispecific antibody willnot effect the release of cytokine. Thus, no cytokine release will occurin parts of the body removed from the tumor and, hence, expression ofELAM-1 will occur only within the tumor endothelium.

A number of useful "cytokine activating" antigens are known, which, whencross-linked with an appropriate bispecific antibody, will result in therelease of cytokines by the cross-linked leukocyte. The most preferredtarget for this purpose is CD14, which is found on the surface ofmonocytes and macrophages. When CD14 is cross linked it will stimulatethe monocyte/ macrophage to release IL-1, and possibly other cytokines,which will, in turn stimulate the appearance of ELAM-1 on nearbyvasculature. Other possible targets for cross-linking in connection withELAM-1 targeting includes FcR for IgE, found on Mast cells; FcR for IgG(CD16), found on NK cells; as well as CD2, CD3 or CD28, found on thesurfaces of T cells. Of these, CD14 targeting will be the most preferreddue to the relative prevalence of monocyte/macrophage infiltration ofsolid tumors as opposed to the other leukocyte cell types.

In that MHC Class II antigens are expressed on "!normal" endothelium,their targeting is not quite so straightforward as ELAM-1. However, thepresent invention takes advantage of the discovery thatimmunosuppressants such as Cyclosporin A (CsA) have the ability toeffectively suppress the expression of Class II molecules in the normaltissues. There are various other cyclosporins related to CsA, includingcyclosporins A, B, C, D, G, and the like, which have immunosuppressiveaction, and will likely also demonstrate an ability to suppress Class IIexpression. Other agents that might be similarly useful include FK506and rapamycin.

Thus, the practice of the MHC Class II targeting embodiment requires apretreatment of the tumor-bearing animal with a dose of CsA or otherClass II immunosuppressive agent that is effective to suppress Class IIexpression. In the case of CsA, this will typically be on the order ofabout 10 to 30 mg/kg. Once suppressed in normal tissues, Class IIantigens can be selectively induced in the tumor endothelium through theuse of a bispecific antibody, this one having specificity for the tumorcell as well as an activating antigen found on the surface of helper Tcells. Note that in this embodiment, it is necessary that T cells, or NKcells if CD16 is used, be present in the tumor to produce the cytokineintermediate in that Class II antigen expression is achieved usingIFN-γ, but is not achieved with the other cytokines. Thus, for thepractice of this aspect of the invention, one will desire to select CD2,CD3 or CD28 (most preferably CD28) as the cytokine activating antigen.

An alternative approach to using "cytokine-activating" bispecificantibodies might be to activate the patients peripheral blood leukocytesor tumor-infiltrating lymphocytes in vitro (using IL-2 or autologoustumor cells for instance), reinfuse them into the patient and thenlocalize them in the tumor with a bispecific antibody against anyreliable leukocyte-specific marker, including CD5, CD8, CD11/CD18, CD15,CD32, CD44, CD45 or CD64. In order to selectively localize thoseleukocytes that had become activated from within a mixed population, itis recommended that the anti-leukocyte arm of the bispecific antibodyshould recognize a marker restricted to activate cells, such as CD25,CD30, CD54 or CD71. Neither of these approaches is favored as much asthe `cytokine-activating` antibody approach because cross-linking totumor cells is not a prerequisite for cytokine secretion and thus theresultant induction of cytokine-induced endothelial cell antigens maynot be confined to the tumor.

The targeting of the other adhesion molecules, ICAM-1, VCAM-1 and LAM-1agent, will typically not be preferred for the practice of therapeuticembodiments, in that these targets are constitutively expressed innormal endothelium. Thus, these adhesion molecules will likely only beuseful in the context of diagnostic embodiments. Furthermore, it isunlikely that ICAM-1 or VCAM-1 expression by normal endothelial cellswould be inhibited in vivo by CsA because low levels of expression ofboth markers are constitutive properties of human endothelial cells(Burrows et al., 1991). However, it may still be possible to utilize oneof these molecules in diagnostic or even therapeutic embodiments becausetheir level of expression on the endothelial cell surface is increased10-50 fold by cytokines. As a consequence, there may be a therapeutic ordiagnostic `window` enabling use of anti-ICAM-1 or anti-VCAM-1conjugates in an analogous way to the proven clinical utility of someantibodies against `tumor-associated` antigens whose expression differsquantitatively but not qualitatively from normal tissues.

The tumor antigen recognized by the bispecific antibodies employed inthe practice of the present invention will be one that is located on thecell surfaces of the tumor being targeted. A large number of solidtumor-associated antigens have now been described in the scientificliterature, and the preparation and use of antibodies are well withinthe skill of the art (see, e.g., Table II hereinbelow). Of course, thetumor antigen that is ultimately selected will depend on the particulartumor to be targeted. Most cell surface tumor targets will only besuitable for imaging purposes, while some will be suitable fortherapeutic application. For therapeutic application, preferred tumorantigens will be TAG 72 or the HER-2 proto-oncogene protein, which areselectively found on the surfaces of many breast, lung and colorectalcancers (Thor et al., 1986; Colcher et al., 1987; Shepard et al., 1991).Other targets that will be particularly preferred include milk mucincore protein, human milk fat globule (Miotti et al., 1985; Burchell etal., 1983) and even the high Mr melanoma antigens recognized by theantibody 9.2.27 (Reisfeld et al., 1982).

In still further embodiments, the inventors contemplate an alternativeapproach for suppressing the expression of Class II molecules, andselectively eliciting Class II molecule expression in the locale of thetumor. This embodiment takes advantage of the fact that the expressionof Class II molecules can be effectively inhibited by suppressing IFN-γproduction by T-cells, e.g., through use of an anti-CD4 antibody (Streetet al., 1989). Thus, in this embodiment, one will desire to pretreatwith a dose of anti-CD4 that is effective to suppress IFN-γ productionand thereby suppress the expression of Class II molecules (for example,on the order of 4 to 10 mg/kg). After Class II expression is suppressed,one will then prepare and introduce into the bloodstream anIFN-γ-producing T-cell clone (e.g., T_(h) 1 or CTL) specific for anantigen expressed on the surface of the tumor cells.

A preferred means of producing the IFN-γ-producing T-cell clone is by amethod that includes removing a portion of the tumor mass from thepatient, extracting tumor infiltrating leukocytes from the tumor, andexpanding the tumor infiltrating leukocytes in vitro to provide theIFN-γ producing clone. This clone will necessarily be immunologicallycompatible with the patient, and therefore should be well tolerated bythe patient. It is proposed that particular benefits will be achieved byfurther selecting a high IFN-γ producing T-cell clone from the expandedleukocytes by determining the cytokine secretion pattern of eachindividual clone every 14 days. To this end, rested clones will bemitogenically or antigenically-stimulated for 24 hours and their culturesupernatants assayed by a specific sandwich ELISA technique (Cherwinskiet al., 1989) for the presence of IL-2, IFN-γ, IL-4, IL-5 and IL-10.Those clones secreting high levels of IL-2 and IFN-γ, the characteristiccytokine secretion pattern of TH. clones, will be selected. Tumorspecificity will be confirmed using proliferation assays. Furthermore,one will prefer to employ as the anti-CD4 antibody an anti-CD4 Fab,because it will be eliminated from the body within 24 hours afterinjection and so will not cause suppression of the tumor recognizing Tcell clones that are subsequently administered. The preparation ofT-cell clones having tumor specificity is generally known in the art, asexemplified by the production and characterization of T cell clones fromlymphocytes infiltrating solid melanoma tumors (Maeda et al., 1991).

The invention contemplates that still further advantages will berealized through combination regimens wherein both the tumor endothelialvasculature and the tumor itself are targeted. Combination regimens maythus include targeting of the tumor directly with either conventionalantitumor therapy, such as with radiotherapy or chemotherapy, or throughthe use of a second immunological reagent such as an antitumorimmunotoxin. In fact, dramatic, synergistic antitumor effects were seenby the inventors when solid tumors were targeted with both an antitumorendothelial cell immunotoxin and an antitumor cell immunotoxin. Suchcombination therapy is founded theoretically on 1) the use of theendothelial-directed immunotoxin to kill those tumor cells that dependupon vascular oxygen and nutrients, and 2) the use of the tumor-directedimmunotoxin to kill those tumor cells that may have an alternate sourceof oxygen and nutrients (i.e., those tumor cells lining the vasculatureand those forming the outer boundary of the tumor mass). Thus, it isproposed that particular advantages will be realized through thetargeting of agents both to tumor cell targets as well as to tumorendothelial cell targets.

The invention further contemplates the selected combinations of agentsparticularly adapted for use in connection with the methods of thepresent invention, defined as including a first pharmaceuticalcomposition which includes a bispecific antibody recognizing anactivating antigen on the cell surface of a leukocyte cell and a tumorantigen on the cell surface of tumor cells of a vascularized solidtumor, together with a second pharmaceutical composition comprising asecond antibody or fragment thereof linked to a selected therapeutic ordiagnostic agent that recognizes the induced endothelial antigen. Inaccordance with one aspect of the invention, these agents may beconveniently packaged together, being suitably aliquoted into separatecontainers, and the separate containers dispensed in a single package.

In particular embodiments, the activating antigen induced by thebispecific antibody will be CD2, CD3, CD14, CD16, FcR for IgE, CD28 orthe T-cell receptor antigen, as may be the case. However, preferably,the bispecific antibody will recognize CD14, and induce the expressionof IL-1 by monocyte/macrophage cells in the tumor, or recognize CD28 andinduce the expression of IFN-γ by T-cells in the tumor. Where IL-1 isthe cytokine intermediate, the second antibody will preferably be onethat recognizes ELAM-1, since this adhesion molecule will be induced onthe surface of endothelial cells by IL-1. In contrast, where IFN-γ isthe intermediate, the second antibody will preferably be one thatrecognizes an MHC Class II antigen. In the later case, one might desireto include with the combination a third pharmaceutical compositioncomprising one of the cyclosporins, or another immunosuppressive agentuseful for suppressing Class II expression.

Furthermore, in that the invention contemplates combination regimens asdiscussed above, particular embodiments of the invention will involvethe inclusion of a third pharmaceutical composition comprising anantitumor antibody conjugated to a selected agent, such as an anti-tumorimmunotoxin. In these embodiments, particularly preferred will be thetargeting of tumor antigens such as p185^(HER2), milk mucin coreprotein, TAG-72, Lewis a, carcinoembryonic antigen (CEA), the high Mrmelanoma antigens recognized by the 9.2.27 antibody, or theovarian-associated antigens recognized by OV-TL3 or MOV18. These sameantigens will also be preferred as the target for the bispecificantibody. Of course, where such a bispecific antibody is employed incombination with an antitumor antibody, it may be desirable to targetdifferent tumor antigens with the bispecific and antitumor antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG, 1a. Induction of I-E^(k) on SVEC cells by IFN-γ in regular medium,r.IFN-γ, or r.IFN-γ plus excess neutralizing anti-IFN-γ antibody. SVECcells were cultured for 72 hours in regular medium ( - - - ), r.IFN-γ (. . . ) or r.IFN-γ plus excess neutralizing anti-IFN-γ antibody ( . . .). Their expression of I-E^(k) was then measured by M5/114 antibodybinding by indirect immunofluorescence using the FACS(fluorescence-activated cell sorter). Other cultures were treated withr.IFN-γ and stained with an isotype-matched control antibody ( - - - ).

FIG, 1b. Induction of I-E^(k) on SVEC cells by IFN-γ inC1300-conditioned media. SVEC cells were cultured for 72 hours inC1300-conditioned medium ( - - - ), C1300(Muγ)-conditioned medium ( . .. ) or C1300(Muγ)-conditioned medium plus excess neutralizing anti-IFN-γantibody ( . . . ). Their expression of I-E^(k) was then measured as inFIG, 1a. Other cultures were treated with C1300(Muγ)-conditioned mediumand stained with an isotype-matched control antibody ( - - - ).

FIG. 2a. Expression of I-E^(k) and H-2K^(k) by pure and mixedpopulations of C1300 and C1300(Muγ) cells stained with anti-I-E^(k)antibody. C1300 cells ( . . . ), C1300(Muγ) cells ( - - - ), a mixtureof C1300 and C1300(Muγ) cells in the ratio 7:3 cocultured in vitro ( . .. ) or cells recovered from a mixed subcutaneous tumor in a BALB/c nu/numouse ( - - - ) were stained with anti-I-E^(k) antibody by indirectimmunofluorescence using the FACS. No staining of any tumor cellpopulation was seen with the isotype-matched control antibodies.

FIG. 2b. Expression of I-E^(k) and H-2K^(k) by pure and mixedpopulations of C1300 and C1300(Muγ) cells stained with anti-H-2K^(k)antibody. C1300 cells ( . . . ), C1300(Muγ) cells (----), a mixture ofC1300 and C1300(Muγ) cells in the ratio 7:3 cocultured in vitro ( . . .) or cells recovered from a mixed subcutaneous tumor in a BALB/c nu/numouse ( - - - ) were stained with anti-H-2K^(k) antibody by indirectimmunofluorescence using the FACS. No staining of any tumor cellpopulation was seen with the isotype-matched control antibodies.

FIG. 3. Tumorigenicity, growth, and tumor endothelial cell Ia^(d)expression in pure and mixed subcutaneous C1300 and C1300(Muγ) tumors.BALB/c nu/nu mice were injected with a total of 2×10⁷ tumor cells inwhich the ratios of C1300: C1300(Muγ) cells were 10:0 (Δ), 9:1 (∘), 8:2(•), 7:3 (⋄), 5:5 (▪), 3:7 (□) or 0:10 (▴). The vertical axis shows themean diameter of the tumors at various times after injection. Also shownare the percentage of animals in each group which developed tumors. Theproportion of Ia^(d) -positive vascular endothelial cells wascategorized as follows: +, 75-100%; +/-, 25-75%; -, 0-5%; n.d., notdetermined because no intact blood vessels were visible. Standarddeviations were <15% of mean diameters and are not shown.

FIG. 4a. Killing activity of anti-Class II immunotoxin (M5/114 dgA)against unstimulated SVEC mouse endothelial cells. The data shown arefor treatment of cells with varying concentrations of ricin (□);M5/114dgA (•); and the control immunotoxin CAMPATH-2dgA (∘).

FIG. 4b. Killing activity of anti-Class II immunotoxin (M5/114 dgA)against SVEC mouse endothelial cells stimulated with conditioned mediumfrom the IFN-γ-secreting tumor C1300(Muγ). The data shown are fortreatment of cells with varying concentrations of ricin (□); M5/114dgA(•); and the control immunotoxin CAMPATH-2dgA (∘).

FIG. 5. This FIG. also shows the killing of SVEC cells under variousconditions by the anti-Class II immunotoxin, M5/114dgA. The data shownare for treatment of cells with varying concentrations of theimmunotoxin following treatment with IFN-γ TCM (∘); C1300 TCM (▴);C1300(Muγ) TCM (▪); and C1300(Muγ) treated with anti-IFN-γ (□).

FIG. 6a. Shows a comparison of killing activity of an anti-Class I(antitumor) immunotoxin (11-4.1-dgA, which recognized H-2K^(k)) and ananti-Class II (anti-tumor endothelial cell) immunotoxin (M5/114-dgA)against a 70:30 mixed population of C1300 and C1300(Muγ) cells. Data wasobtained through treatment of the cells with ricin (•); the 11-4.1-dgAimmunotoxin (∘); the M5/114-dgA immunotoxin (▪) and a controlimmunotoxin (□).

FIG. 6b. Shows killing of cells freshly recovered from subcutaneoustumors in mice. Data was obtained through treatment of the cells withricin (•); the 11-4.1-dgA immunotoxin (∘); the M5/114-dgA immunotoxin(▪) and a control immunotoxin (□).

FIG. 7a. Killing of pure populations of C1300 (•) and C1300(Muγ) (∘) bythe antitumor cell immunotoxin, 11-4.1-dgA. Also shown are 70:30 mixedpopulations mixed in vitro or in vivo (i.e., recovered from S/C tumors).Also shown are controls, including ricin (▴) and a control immunotoxin(Δ).

FIG. 7b. Killing of pure populations of C1300 (•) and C1300(Muγ) (∘) bythe antitumor cell immunotoxin, 11-4.1-dgA, as shown in FIG. 7a, withthe controls, ricin (▴) and a control immunotoxin (Δ), for comparison.

FIG. 8. This FIG. shows the in vivo antitumor effects of theanti-endothelial cell immunotoxin, M5/114-dgA, at various doses,including 20 μg (∘) and 40 μg (▪). These studies involved theadministration of the immunotoxin intravenously 14 days after injectionof tumor cells. Controls included the use of a control immunotoxin,CAMPATH-2-dgA (Δ) and PBS +BSA (▴).

FIG. 9. This FIG. is a histological analysis of 1.2 cm H&E-stained tumorsections 72 hours after treatment with 20 μg of the anti-Class IIimmunotoxin, M5/114-dgA.

FIG. 10. This FIG. is a histological analysis of 1.2 cm H&E-stainedtumor sections 72 hours after treatment with 100 μg of the anti-Class IIimmunotoxin, M5/114-dgA.

FIG. 11. This FIG. is a representation of the appearance of a solidtumor 48-72 hours after intravenous immunotoxin treatment, and comparesthe effect achieved with anti-tumor immunotoxin, to that achieved withanti-endothelial cell immunotoxin therapy.

FIG. 12. This FIG. shows the antitumor effects of single and combinedtreatments with anti-Class I and anti-Class II immunotoxins in SCID micebearing large solid C1300(Muγ) tumors. SCID mice bearing 1.0-1.3 cmdiameter tumors were injected intravenously 14 days after tumorinoculation with 20 μg of Class II immunotoxin (∘), 100 μg Class Iimmunotoxin (•), or diluent alone (▴). Other animals received theanti-Class II immunotoxin followed by two days later by the anti-Class Iimmunotoxin (▪), or vice versa (□). Tumor size was measured at regularintervals and is expressed as mean tumor diameter +/- SEM. Eachtreatment group consisted of 4-8 mice.

FIG. 13a. Gel electrophoretic analysis of proteins immunoprecipitatedfrom ³⁵ S-labelled human umbilical vein endothelial cells (HUVEC),showing that TEC-4 and TEC-11 recognize endoglin. 12.5% SDS-PAGE gel ofproteins immunoprecipitated under reducing (lanes 2-4) or non-reducing(lanes 5-7) conditions with TEC-4 (lanes 2,5), TEC-11 (lanes 3,6) orTEPC-183 (lanes 4,7). Lane 1: Position of the ¹⁴ C-labelled standards ofthe molecular weights indicated. Lane 8: Positions of 95 kDa and 180 kDaspecies.

FIG. 13b. Reactivity of TEC-4 and TEC-11 with human endoglintransfectants, showing that TEC-4 and TEC-11 recognize endoglin.Parental murine L cells and L cell transfectants expressing humanendoglin were incubated with purified MAb TEC-11, TEC-4 and 44G4followed by FITC-conjugated F(ab')₂ goat anti-mouse IgG (H+L). Thestaining observed on the parental L cells with the MAb (whitehistograms) was indistinguishable from that observed with IgM and IgG1controls. The L cell endoglin transfectants (black histograms) werespecifically reactive with all 3 antibodies as revealed by thepercentage of cells included within the gate and shown in parentheses.

FIG. 14. Crossblocking of TEC-4 and TEC-11 antibodies. Biotinylatedantibodies (10 μg/ml) were mixed with an equal volume of unlabelledTEC-4 antibody at 10 μg/ml (□), 100μg/ml or 1000 μg/ml (▪) or withunlabelled TEC-11 antibody at 10 μg/ml 100 μg/ml and were added to HUVECin PBS-BSA-N₃. Indirect immunofluorescence staining was carried out asdescribed in Example V with the exception that labelled antibody bindingwas detected with a streptavidin-phycoerythrin conjugate. Each group ofhistograms shows the percent blocking of the biotinylated antibody bythe different concentrations of unlabelled antibodies. Bar: SD oftriplicate determinations.

FIG. 15. Complement fixation by TEC-4 and TEC-11 antibodies. HUVEC wereincubated with TEC-4 (), TEC-11 (▪) or MTSA (▴) antibodies, washed andsubsequently incubated with guinea-pig complement. Cell number andviability were determined by trypan blue dye exclusion.

FIG. 16a. Correlation between TEC-11 binding and cellular proliferationin HUVEC. HUVEC from sparse cultures (hatched histogram) orpost-confluent cultures (open histogram) were stained with TEC-11 byindirect immunofluorescence. Also shown, confluent HUVEC stained withnegative control antibody MTSA (stippled histogram). Endoglin^(lo) andendoglin^(hi) populations of post-confluent HUVEC were separated on aFACStar Plus cell sorter as indicated and subsequently analyzed for RNAand DNA content.

FIG. 16b. Lack of correlation between LM142 binding and cellularproliferation in HUVEC. HUVEC from sparse (hatched histogram) orpost-confluent (open histogram) were stained with the anti-vitronectinreceptor antibody LM142. Also shown, sparse HUVEC stained with negativecontrol antibody MTSA (stippled histogram).

FIG. 16c. Correlation between TEC-11 binding and cellular proliferationin HUVEC. Endoglin^(lo) HUVEC assayed for acridine orange interrelationinto cellular DNA (x-axes) and DNA (y-axes). Essentially all cellscontained low levels of RNA and DNA and were located within the lowerleft (G₀) quadrant.

FIG. 16d. Correlation between TEC-11 binding and cellular proliferationin HUVEC. Endoglin^(hi) HUVEC assayed for RNA and DNA as described inthe above legend. Significant numbers of cells contain increased RNAlevels (G₁ phase, lower right quadrant) and increased RNA and DNA levels(S+G₂ M phases, upper right quadrant).

FIG. 17a. Immunohistochemical detection of TEC-4 binding to malignanthuman parotid tumor tissue. Numerous blood vessels are stained stronglyby TEC-4 in a parotid tumor whereas only a single stained vessel ispresent in the adjacent normal glandular tissue (b,arrow). Antibodybinding was detected with biotinylated F(ab')₂ rabbit anti-mouse Ig andSABC-HRP with AEC substrate and hematoxylin counterstaining; ×40.

FIG. 17b. Immunohistochemical detection of TEC-4 binding to normal humanglandular tissue. Only a single vessel is stained by TEC-4 in thisnormal glandular tissue (b,arrow). Antibody binding was detected withbiotinylated F(ab')₂ rabbit anti-mouse Ig and SABC-HRP with AECsubstrate and hematoxylin counterstaining; ×40.

FIG. 17c. Immunohistochemical detection of TEC-4 binding to human breastcarcinoma tissues. Under high power, endothelial cells in a breastcarcinoma are strongly stained with TEC-4 whereas normal umbilical veinendothelial cells are weak-negative. Antibody binding was detected withbiotinylated F(ab')₂ rabbit anti-mouse Ig and SABC-HRP with AECsubstrate and hematoxylin counterstaining; ×64.

FIG. 17d. Immunohistochemical detection of TEC-4 binding to normalumbilical vein endothelial cells. Antibody binding was detected withbiotinylated F(ab')₂ rabbit anti-mouse Ig and SADC-HRP with AECsubstrate and hematoxylin counterstaining; ×20.

FIG. 18a. Differential TEC-4 binding to endothelial cells in normalbreast tissue, control. Sections of normal mammary gland were stainedwith a control anti-endothelial cell antibody, F8/86 (anti-vonWillebrands Factor). Antibody binding was detected as in the legend toFIG. 16 except that DAB substrate was used and a light hematoxylincounterstaining was applied; ×20.

FIG. 18b. Differential TEC-4 binding to endothelial cells in normalbreast tissue, TEC-4 control. Sections of normal mammary gland werestained with TEC-4. Binding of TEC-4 to normal endothelial cells is notseen. Antibody binding was detected as in the legend to FIG. 16 exceptthat DAB substrate was used and a light hematoxylin counterstaining wasapplied; ×20.

FIG. 18c. Differential TEC-4 binding to endothelial cells in malignantbreast tissue, control. Sections of breast carcinoma were stained with acontrol anti-endothelial cell antibody, F8/86 (anti-von WillebrandsFactor). Antibody binding was detected as in the legend to FIG. 16except that DAB substrate was used and a light hematoxylincounterstaining was applied; ×20.

FIG. 18d. Differential TEC-4 binding to endothelial cells in malignantbreast tissue, TEC-4. Sections of breast carcinoma were stained withTEC-4. Binding of TEC-4 to endothelial cells is only seen in themalignant breast sample. Antibody binding was detected as in the legendto FIG. 16 except that DAB substrate was used and a light hematoxylincounterstaining was applied; ×20.

FIG. 19a. No significant killing of proliferating HUVEC by MTSA-dgA.Quiescent (•), confluent (▪) or subconfluent (▴) HUVEC cultures wereincubated for 48 hours with a negative control immunotoxin (MTSA-dgA).Protein synthesis was estimated from the uptake of ³ H-leucine duringthe last 24 hours of culture. Points and bars: mean and standard errorof 6 individual studies.

FIG. 19b. Significant killing of proliferating HUVEC by UV- 3-dgA.Quiescent (•), confluent (▪) or subconfluent (▴) HUVEC cultures wereincubated for 48 hours with a positive control immunotoxin (UV-3-dgA).Protein synthesis was estimated from the uptake of ³ H-leucine duringthe last 24 hours of culture. Points and bars: mean and standard errorof 6 individual studies.

FIG. 19c. Significant killing of proliferating HUVEC by ricin. Quiescent(•), confluent (▪) or subconfluent (▴) HUVEC cultures were incubated for48 hours with ricin. Protein synthesis was estimated from the uptake of³ H-leucine during the last 24 hours of culture. Points and bars: meanand standard error of 6 individual studies.

FIG. 19d. Significant killing of proliferating HUVEC by TEC-11dgA.Quiescent (•), confluent (▪) or subconfluent (▴) HUVEC cultures wereincubated for 48 hours with TEC-11dgA. Protein synthesis was estimatedfrom the uptake of ³ H-leucine during the last 24 hours of culture.Points and bars: mean and standard error of 6 individual studies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although they show great promise in the therapy of lymphomas andleukemias (Lowder et al., 1987; Vitetta et al., 1991), monoclonalantibodies (mAbs), immunotoxins (ITs) and other immunoconjugates havethus far proved relatively ineffective in clinical trials againstcarcinomas and other solid tumors (Byers and Baldwin, 1988; Abrams andOldham, 1985), which account for more than 90% of all cancers in man(Shockley et al., 1991). A principal reason for this is thatmacromolecules do not readily extravasate into solid tumors (Sands,1988; Epenetos et al., 1986) and, once within the tumor mass, fail todistribute evenly due to the presence of tight junctions between tumorcells (Dvorak et al., 1991), fibrous stroma (Baxter and Jain, 1991),interstitial pressure gradients (Jain, 1990) and binding site barriers(Juweid et al., 1992).

A solution to the problem of poor penetration of antibodies into solidtumors is to attack the endothelial cells (EC) lining the blood vesselsin the tumor. This approach offers several advantages over directtargeting of tumor cells. Firstly, the target cells are directlyaccessible to intravenously administered therapeutic agents, permittingrapid localization of a high percentage of the injected dose (Burrows etal., 1990; Kennel, et al., 1991). Secondly, since each capillaryprovides oxygen and nutrients for thousands of cells in its surrounding`cord` of tumor, even limited damage to the tumor vasculature couldproduce an avalanche of tumor cell death (Denekamp, 1984; 1986; 1990;Burrows and Thorpe, 1993). The outgrowth of mutant endothelial cellslacking the target antigen is unlikely because endothelial cells arenormal and not neoplastic cells. Finally, endothelial cells are similarin different tumors, making it feasible to develop a single reagent fortreating numerous types of cancer.

For tumor vascular targeting to succeed, targeting agents, such asantibodies, are required that recognize tumor endothelial cells but notthose in normal tissues. Differences between tumor blood vessels andnormal ones have been documented (reviewed in Denekamp, 1990; Dvorak etal., 1991; and Jain, 1988) which suggested to the inventors thatantigenic differences might exist. For example, tumors elaborateangiogenic factors (Kandel et al., 1991; Folkman, 1985) and cytokines(Burrows et al., 1991; Ruco et al., 1990; Borden et al., 1990) whichalter the behavior and phenotype of local endothelial cells. Vascularendothelial cells in tumors proliferate at a rate 30-fold greater thanthose in miscellaneous normal tissues (Denekamp et al., 1982),suggesting that proliferation-linked determinants could serve as markersfor tumor vascular endothelial cells.

Nevertheless, despite fairly intensive efforts in several laboratories(Duijvestijn et al., 1987, Hagemeier et al., 1986; Schlingmann et al.,1985), antibodies have not yet been obtained which clearly distinguishtumor from normal vasculature. Migration-linked endothelial markers havebeen described, but as yet none has been found to be reliably andselectively expressed in the tumor vasculature (Gerlach et al., 1989;Hagemeier et al., 1986; Sarma et al., 1992). For example, the antigenrecognized by the antibody termed EN7/44 (Hagemeier et al., 1986)appears to be linked to migration rather than to proliferation, and,since it is almost entirely cytoplasmically located, is not believed tobe a good candidate for tumor vasculature targeting.

VEGF receptors ar known to be upregulated on tumor endothelial cells asopposed to endothelial cells in normal tissues, both in rodents and man.Possibly, this is a consequence of hypoxia--a characteristic of thetumor microenvironment. FGI receptors are also upregulated three-fold onendothelial cells exposed to hypoxia, and so are probably upregulated intumors.

Both VEGF and bFGF are concentrated in or on tumor vasculature andpotentially provide a target for attack on tumor vasculature. TGF βreceptor (endoglin) on endothelial cells is upregulated on dividingcells (as shown herein), explaining the greater binding on TEC-11 totumor vasculature where endothelial cells are dividing.

Tumor endothelial markers could potentially be induced directly bytumor-derived cytokines (Burrows et al., 1991; Ruco et al., 1990) orangiogenic factors (Mignatti et al., 1991). In support of the existenceof tumor vasculature markers, two antibodies against unrelated antigensof unknown function that are expressed in the vasculature of humantumors but not in most normal tissues have been described subsequent tothe present invention (Rettig et al., 1992; Wang et al., 1993).

The present inventors have developed a variety of strategies forspecifically targeting the targeting agents, such as antibodies, totumor vasculature, which strategies address the shortcomings in theprior approaches. One strategy for vascular targeting presented by theinvention involves the use of a targeting agent, such as an antibodydirected against a tumor vasculature-associated antigen, whetherspecifically bound to the vasculature surface or expressed by anendothelial cell, to target or deliver a selected therapeutic ordiagnostic agent to the tumor. In addition to an antibody, the targetingagent may be a growth factor which specifically binds a growth factorreceptor present on the surface of a tumor-associated endothelial cell.A second approach involves the selective induction of MHC Class IImolecules on the surfaces of tumor-associated endothelia which can thenserve as endothelial cell targets. A third, related but distinctapproach involves the selective elicitation of an endothelial marker inthe tumor vascular endothelium and the targeting of such as antigen withan appropriate antibody. Naturally, the existence of endothelialmarkers, such as ELAM-1, VCAM-1, ICAM-1 and the like, has beendocumented, however exploiting such molecules by selective induction andsubsequent targeting has not been described previously.

A. IDENTIFICATION OF EXISTING TUMOR VASCULATURE MARKERS

The present inventors developed a novel approach to identify tumorvascular antigens which employs tumor-conditioned cell culture media toinduce specific antigens on vascular endothelial cells. The conditionedmedia, which undoubtedly includes numerous cytokines, growth factors andtumor-specific products, mimics the solid tumor vascular environment andthereby promotes the appearance of specific tumor vascular antigenmarkers. This approach allows specific markers of tumor vasculature tobe identified and then targeted with a targeting agent, such as aspecific antibody, linked, or operatively attached to, a selectedtherapeutic or diagnostic agent.

The generation of antibodies against specific markers of tumorvasculature generally involves using the stimulated endothelial cells asimmunogens in an animal system. The methods of generating polyclonalantibodies in this manner are well known, as are the techniques ofpreparing monoclonal antibodies via standard hybridoma technology. Theinventors also contemplate the use of a molecular cloning approach togenerate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning on normal-versus-tumor endothelium. The advantagesof this approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination which further increases the chance of findingappropriate antibodies.

In one of the studies disclosed herein, the inventors report thedevelopment and characterization of two murine IgM monoclonalantibodies, TEC-4 and TEC-11, which are envisioned to be suitable fortargeting the tumor vasculature of humans. TEC-4 and TEC-11 wereinitially believed to recognize an antigen that migrated as a 43 kDdoublet on SDS/PAGE. However, as detailed herein, the present inventorssubsequently determined this antigen to be the molecule endoglin, whichis a dimeric glycoprotein consisting of 95 kDa disulfide-linked subunitswhose primary sequence is known (Gougos, 1990). Monoclonal antibodieshave previously been raised against endoglin (Gougos and Letarte, 1988;Gougos et al., 1992; O'Connel et al., 1992; Buhring et al., 1991).However, TEC-4 and TEC-11 are believed to recognize distinct epitopes,as shown, for example, by the failure of 44G4 to block TEC-4 or TEC-11,even at high ratios.

Endoglin is expressed on human endothelial cells, fetalsyncytiotrophoblast (Gougos et al., 1992), some macrophages (O'Connellet al., 1992), immature erythroid cells (Euhring et al., 1991), and someleukemic and hemopoietic cell lines (Gougos and Letarte, 1988; Gougos etal., 1992; O'Connel et al., 1992; Buhring et al., 1991). Its expressionon dermal endothelium has recently been demonstrated to be upregulatedin several chronic inflammatory skin lesions (Westphal et al., 1993).

Using the TEC-4 and TEC-11 antibodies, the inventors report herein thatendoglin is upregulated on activated and dividing HUVEC in culture, andis strongly expressed in human tissues on endothelial cells at sites ofneovascularization, including a broad range of solid tumors and fetalplacenta. In contrast, endothelial cells in the majority ofmiscellaneous non-malignant adult tissues, including preneoplasticlesions, were only weakly positive or not stained by TEC-4 and TEC-11.Importantly, TEC-4 and TEC-11 antibody binding is also shown tocorrelate with neoplastic progression in the breast: benignfibroadenomas, and early carcinoma-in-situ bound low levels of TEC-4 andTEC-11 whereas late stage intraductal carcinomas and invasive carcinomasbound high levels of the antibodies.

HUVEC in sections of umbilical vein react weakly with TEC-4 and TEC-11,whereas proliferating HUVEC in tissue culture react strongly anduniformly. HUVEC cultures grown to confluence and then rested containtwo subpopulations having high and low levels of endoglin expression.Multiparameter analysis by FACS revealed that a significant proportionof cells with high endoglin-expression are cycling, having markedlyincreased levels of cellular protein, RNA and DNA by comparison with lowendoglin-expressing cells, which appear all to be non-cycling. Takentogether, the increased binding of TEC-4 and TEC-11 to tumor vasculatureand to dividing as opposed to non-cycling HUVEC in vitro indicates thatendoglin is an endothelial cell proliferation-associated marker.Anti-endoglin antibodies are thus proposed to have broad-basedapplicability in the diagnosis, imaging and therapy of solid tumors inman. Furthermore, the uniformity of staining of vessels in differenttumors and within any individual tumor indicates that TEC-4 and TEC-11compare favorably with various other antibodies, such as, e.g., FB-5(Rettig et al., 1992) and E9 (Wang et al., 1993).

An additional concept of one of the present inventors is the use of"tumor-derived endothelial cell binding factors" as a means ofdistinguishing between tumor vasculature and the vasculature of normaltissues. That is, if tumors secrete factors for which vascularendothelial cells have receptors, the endothelial cells in the tumorwill capture that factor and display it on their surface. In contrast,endothelial cells in normal tissues will bind relatively little of thefactor because it is diluted within the blood pool or because thereceptors on normal endothelial cells are not upregulated as they are ontumor endothelial cells. Thus, operationally, the tumor-derived factorwill also constitute a tumor endothelial cell marker.

Such tumor-derived endothelial cell binding factors can be manufacturedby the tumor cells themselves, by cells (e.g. macrophages, mast cells)which have infiltrated tumors or by platelets which become activatedwithin the tumor. It is proposed that an antibody or other ligand whichrecognizes that factor will home selectively to tumor vasculature afterinjection. Such an antibody or ligand should thus enable the imaging ortargeting of drugs or other agents to solid tumors. Further, such anantibody may be specific for a factor/factor receptor complex present onthe surface of the tumor vasculature, so that the antibody recognizesonly a factor/factor receptor complex, while not binding to either thefactor or the factor receptor individually.

Various candidate factors include, for example, vascular endothelialcell growth factor (VEGF), also called vascular permeability factor(VPF); members of the fibroblast growth factor (FGF) family, e.g., basicFGF; Tumor necrosis factor-α (TNF-α); transforming growth factor-α(TGF-α) and transforming growth factor-β (TGF-β); angiogenic;angiotropin; and platelet-derived endothelial cell growth factor(PD-ECGF).

B. SELECTIVE INDUCTION OF OTHER TUMOR VASCULATURE MARKERS

Another approach to targeting tumor vasculature involves the selectiveinduction of a molecule that is capable of acting as a marker forsubsequent tumor endothelial cell targeting. Within this generalstrategy, the inventors have focused on both the induction of MHC ClassII molecules and the induction of endothelial cell adhesion molecules.The MHC Class II approach, however, requires that MHC Class IIexpression be effectively inhibited in normal tissues. It is known thatCsA and related immunosuppressants have this capability via inhibitionof T cell activation, and can therefore be employed to pretreat thepatient or animal to inhibit Class II expression. Alternatively, it isproposed that inhibition of Class II expression can be achieved usinganti-CD4 in that CD4 directed antibodies are known to additionallysuppress T cell function (Street et al., 1989). Then, Class II targetsare selectively induced in the tumor-associated vascular endotheliumthrough a locally released cytokine intermediate (IFN-γ).

To use the related approach of selectively eliciting an endothelialmarker in tumor vascular endothelium, one may exploit one or more of thevarious endothelial adhesion molecules. The expression of an endothelialadhesion molecule, such as ELAM-1, VCAM-1, ICAM-1, LAM-1 ligand etc.,may thus be selectively induced and then targeted with an appropriateantibody. Of these, ELAM-1 is the preferred target in that it is quiteclear that this antigen is not expressed in normal endothelialvasculature (Cotran et al., 1986). The other adhesion molecules appearto be expressed to varying degrees in other normal tissues, generally inlymphoid organs and on endothelium, making their targeting perhapsappropriate only in diagnostic embodiments.

In either case, the key is the use of a bispecific "cytokine-inducing"antibody that will selectively induce the release of the appropriatecytokine in the locale of the tumor. This specifically localized releaseof cytokine is achieved through a bispecific antibody having the abilityto "cross-link" cytokine-producing leukocytes to cells of the tumormass. The preparation and use of bispecific antibodies such as these ispredicated in part on the fact that cross-linking antibodies recognizingCD3, CD14, CD16 and CD28 have previously been shown to elicit cytokineproduction selectively upon cross-linking with the second antigen (Qianet al., 1991). In the context of the present invention, since onlysuccessfully tumor cell-crosslinked leukocytes will be activated torelease the cytokine, cytokine release will be restricted to the localeof the tumor. Thus, expression of ELAM-1 will be similarly limited tothe endothelium of the tumor vasculature.

An overview of various exemplary inducible vascular endothelial targets,as well as the mechanisms for their induction, is set forth in Table I.This Table lists various potential endothelial cell targets, such asELAM-1, VCAM-1, etc., the inducing intermediate cytokine, such as IL-1,IFN-γ, etc., and the leukocyte cell type and associated cytokineactivating molecule whose targeting will result in the release of thecytokine. Thus, for example, a bispecific antibody targeted to anappropriate solid tumor antigen and CD14, will promote the release ofIL-1 by tumor-localized monocytes and macrophages, resulting in theselective expression of the various adhesion molecules in the tumorvascular endothelium. Alternatively, the bispecific antibody may betargeted to FcR for IgE, FcR for IgG (CD16), CD2, CD3, or CD28, andachieve a similar result, with the cytokine intermediate andcytokine-producing leukocyte being different or the same.

                                      TABLE I                                     __________________________________________________________________________    POSSIBLE INDUCIBLE VASCULAR TARGETS                                                                                         LEUKOCYTE MOLECULES WHICH,                                                          WHEN CROSSLINKED BY                                                      INDUCIBLE  SUBTYPES/ALIASES                                                    LEUKOCYTES WHICH MONOCLONA                                                  L ANTIBODIES                      ENDOTHELIAL  (MOLECULAR INDUCING PRODUCE THOSE ACTIVATE THE CELLS TO                                                       CELL MOLECULES ACRONYM                                                       FAMILY) CYTOKINES CYTOKINES                                                   PRODUCE CYTOKINES               __________________________________________________________________________    Endothelial-                                                                             ELAM-1                                                                              --         IL-1, TNF-                                                                           monocytes  CD14                              Leukocyte  (Selectin) α, (TNF-β) macrophages CD14                  Adhesion   (Bacterial mast cells FcR for IgE                                  Molecule-1   Endotoxin)                                                       Vascular Cell VCAM-1 Inducible Cell (Bacterial monocytes CD14                 Adhesion  Adhesion Endotoxin) macrophages CD14                                Molecule-1  Molecule-110 IL-1, TNF- mast cells FcR for IgE                      (INCAM-110) α                                                           (Immunoglobulin TNF-β, IL- helper T cells CD2, CD3, CD28                                                              Family) 4                         TNF NK cells FcR for IgG (CD16)                                            Intercellular ICAM-1 -- IL-1, TNFα monocytes CD14                       Adhesion  (Immunoglobulin (Bacterial macrophages CD15                         Molecule-1  Family) Endotoxin) mast cells FcR for IgE                            TNF-β, T helper cells CD2, CD3, CD28                                     IFNγ NK cells FcR for IgG (CD16)                                     The Agent for LAM-1 MEL-14 Agent Il-1, TNFα monocytes CD14                                                           Leukocyte Agent (Mouse)                                                      (Bacterial macrophages CD14       Adhesion   Endotoxin) mast cells FcR for IgE                                  Molecule-1                                                                    Major MHC HLA-DR    Human IFN-γ helper T cells CD2, CD3, CD28                                                        histocompatibility Class                                                     HLA-DP  NK cells FcR for                                                      IgG (CD16)                        Complex II HLA-DQ                                                             Class II  I-A    Mouse  NK cells FcR for IgG (CD16)                           Antigen  I-E                                                                __________________________________________________________________________

As pointed out, the distinction between the selective activation ofELAM-1 and the MHC Class II molecules rests on the fact that ELAM-1 isnot normally expressed in normal epithelium, whereas Class II moleculesare normally expressed in normal endothelium. Thus, when one seeks totarget MHC Class II antigens, it will be important to first inhibittheir expression in normal tissues using CsA or a similarimmunosuppressant agent having the ability to suppress MHC Class IIexpression. Then, MHC Class II molecules can be selectively induced inthe tumor vasculature using, e.g., a bispecific antibody against a solidtumor antigen that activates Thl cells in the tumor in a CsA-independentfashion, such as CD28. Such an antibody will trigger the release ofIFN-γ which, in turn, will result in the selective expression of ClassII molecules in the tumor vasculature.

An alternative approach that avoids both the use of CsA and a bispecificactivating antibody involves the use of anti-CD4 to suppress IFN-γproduction, followed by introduction of an IFN-γ-producing T-cell clone(e.g., T_(h) 1 or cytotoxic T-lymphocytes (CTLs)) that is specific for aselected tumor antigen. In this embodiment, the T-cell clone itselflocalizes to the tumor mass due to its antigen recognition capability,and only upon such recognition will the T-cell clone release IFN-γ. Inthis manner, cytokine release is again restricted to the tumor, thuslimiting the expression of Class II molecules to the tumor vasculature.

T lymphocytes from the peripheral blood (Mazzocchi et al., 1990) orwithin the tumor mass (Fox et al., 1990) will be isolated by collagenasedigestion where necessary, and density gradient centrifugation followedby depletion of other leukocyte subsets by treatment with specificantibodies and complement. In addition, CD4⁺ or CD8 T cell subsets maybe further isolated by treatment with anti-CD8 or anti-CD4 andcomplement, respectively. The remaining cells will be plated at limitingdilution numbers in 96-well (round bottom) plates, in the presence of2×10⁵ irradiated (2500 rad) tumor cells per well. Irradiated syngeneiclymphocytes (2×10⁵ per well) and interleukin-2 (10 U/ml) will also beincluded. Generally, clones can be identified after 14 days of in vitroculture. The cytokine secretion pattern of each individual clone will bedetermined every 14 days. To this end, rested clones will bemitogenically or antigenically-stimulated for 24 hours and their culturesupernatants assayed for the presence of IL-2, IFN-γ, IL-4, IL-5 andIL-10. Those clones secreting high levels of IL-2 and IFN-γ, thecharacteristic cytokine secretion pattern of T_(H1) clones, will beselected. Tumor specificity will be confirmed utilizing proliferationassays.

Supernatants obtained after 24 hour mitogen or antigen-stimulation willbe analyzed in the following cytokine assays: IL-2, IFN-γ, IL-4, IL-5and IL-10. The levels of IL-2 and IL-4 will be assayed using the HT-2bioassay in the presence of either anti-IL-2, anti-IL-4 antibodies orboth. The remaining cytokines will be assayed using specific two-sitesandwich ELISAs (Cherwinski et al., 1989). Cytokines in the unknownsamples will be quantitated by comparison with standard curves, by usingeither linear or four-parameter curve-fitting programs.

A few generalizations can be made as to which approach would be the moreappropriate for a given solid tumor type. Generally speaking, the more"immunogenic" tumors would be more suitable for the MHC Class IIapproach involving, e.g., the cross-linking of T-cells in the tumorthrough an anti-CD28/anti-tumor bispecific antibody, because thesetumors are more likely to be infiltrated by T cells, a prerequisite.Examples of immunogenic solid tumors include renal carcinomas,melanomas, a minority of breast and colon cancers, as well as possiblypancreatic, gastric, liver, lung and glial tumor cancers. These tumorsare referred to as "immunogenic" because there is evidence that theyelicit immune responses in the host and they have been found to beamenable to cellular immunotherapy (Yamaue et al., 1990). In the case ofmelanomas and large bowel cancers, the most preferred antibodies for usein these instances would be B72.3 (anti-TAG-72) and PRSCS/PR4C2(anti-Lewis a) or 9.2.27 (anti-high Mr melanoma antigen).

For the majority of solid tumors of all origins, an anti-CD14 approachthat employs a macrophage/monocyte intermediate would be more suitable.This is because most tumors are rich in macrophages. Examples ofmacrophage-rich tumors include most breast, colon and lung carcinomas.Examples of preferred anti-tumor antibodies for use in these instanceswould be anti-HER-2, B72.3, SM-3, HMFG-2, and SWA11 (Smith et al.,1989).

The inventors have recently developed a model system in the mouse inwhich to demonstrate and investigate immunotoxin-mediated targeting ofvascular endothelial cells in solid tumors. A neuroblastoma transfectedwith the murine interferon-γ (IFN-65 ) is grown in SCID or BALB/c nudemice reared under germ-free conditions. The IFN-γ secreted by the tumorcells induces the expression of MHC Class II antigens on the vascularendothelial cells in the tumor. Class II antigens are absent from thevasculature of normal tissues in germ-free SCID and nude mice althoughthey are present on certain non-life-sustaining normal cells (such as onB lymphocytes and monocytes) and some epithelial cells.

When mice with large (1.2 cm diameter) tumors were injected withanti-Class II-ricin A chain immunotoxin, dramatic anti-tumor effectswere observed. Histological examination of tumors taken from mice atvarious times after injecting the immunotoxin revealed that vascularendothelial cell degeneration was the first discernable event followedby platelet deposition on the injured vessels and coagulation of thetumor blood supply. This was followed by extensive tumor celldegeneration which occurred within 24 hours after injection of theimmunotoxin. By 72 hours, no viable tumor cells remained apart from afew cells on the edge of the tumor where it penetrated into the normaltissues. These surviving tumor cells could be killed by administering animmunotoxin directed against the tumor cells themselves, resulting inlasting complete tumor regressions in a third of the animals.

These background studies have demonstrated the feasibility of targetingtumor vasculature through targeting of MHC Class II or adhesionmolecules such as ELAM-1.

C. MHC CLASS II

Class II antigens are expressed on vascular endothelial cells in mostnormal tissues in several species, including man. Studies in vitro(Collins et al., 1984; Daar et al., 1984; O'Connell et al., 1990) and invivo (Groenewegen et al., 1985) have shown that the expression of ClassII antigens by vascular endothelial cells requires the continuouspresence of IFN-γ which is elaborated by T_(H1) cells and, to a lesserextent, by NK cells and CD8⁺ T cells. As shown in the dog (Groenewegenet al., 1985) and as confirmed by the inventors in normal mice, Class IIexpression through the vasculature is abolished when CsA isadministered. The CsA acts by preventing the activation of T cells andNK cells (Groenewegen et al., 1985; DeFranco, 1991), thereby reducingthe basal levels of IFN-γ below those needed to maintain Class IIexpression on endothelium.

A strategy for confining Class II expression to tumor vasculature is tosuppress IFN-γ production through out the animal by administering CsAand then to induce IFN-γ production specifically in the tumor bytargeting a CsA-resistant T cell activator to the tumor. A bispecific(Fab'-Fab') antibody having one arm directed against a tumor antigen andthe other arm directed against CD28 should localize in the tumor andthen crosslink CD28 antigens on T cells in the tumor. Crosslinking ofCD28, combined with a second signal (provided, for example, by IL-1which is commonly secreted by tumor cells (Burrows et al., 1991; Ruco etal., 1990) has been shown to activate T cells through a CA²⁺-independent non-CsA-inhibitable pathway (Hess et al., 1991; June etal., 1987; Bjorndahl et al., 1989). The T cells that should be activatedin the tumor are those adjacent to the vasculature since this is theregion most accessible to cells and is also where the bispecificantibody will be most concentrated. The activated T cells should thensecrete IFN-γ which induces Class II antigens on the adjacent tumorvasculature.

MHC Class II antigens are not unique to vascular endothelial cells. Theyare expressed constitutively on B cells, activated T cells, cells ofmonocyte/macrophage linage and on certain epithelial cells both in mice(Hammerling, 1976) and in man (Daar et al., 1984). It would therefore beanticipated that damage to these normal tissues would result ifanti-Class II immunotoxin were to be administered. However thispresumption is not correct, at least in mice. Anti-Class II immunotoxinsadministered intravenously to germ-free SCID or BALB/c nude mice are nomore toxic to the mice than are immunotoxins having no reactivity withmouse tissues. There are a number of possible explanations for thissurprising result. First, anti-Class II antibodies injectedintravenously do not appear to reach the epithelial cells or themonocytes/macrophages in organs other than the liver and spleen.Presumably this is because the vascular endothelium in most organs istight, not fenestrated as it is in the liver and spleen, and so theantibodies must diffuse across basement membranes to reach the ClassIi-positive cells.

Secondly, hepatic Kupffer cells and probably other cells ofmonocyte/macrophage lineage are not killed by the anti-Class IIimmunotoxin even though it binds to them. No morphological changes inthe Kupffer cells are visible even several days after administration ofthe immunotoxin. This is probably because cells of monocyte/macrophagelinage are generally resistant to immunotoxin-mediated killing (Engertet al., 1991). Cells of monocyte/macrophage lineage appear to bind andinternalize immunotoxins but route them to the lysosomes where they aredestroyed, unlike other cell types which route immunotoxins to thetrans-Golgi region or the E.R. which are thought to be site(s) fromwhich ricin A chain enters the cytosol (Van Deurs et al., 1986; VanDeurs et al., 1988).

Finally, there were little morphological evidence of splenic damagedespite the fact that the immunotoxin bound to the B cells and that thecells are sensitive to anti-Class II immunotoxins (Lowe et al., 1986; Wuet al., 1990). It is possible that the B cells were killed, but, beingmetabolically inactive, they degenerated very slowly. In any event, Bcell elimination is unlikely to be a significant problem in mice or inman because the cells would be replenished from Class II negativeprogenitors (Lowe et al., 1986); indeed, in B lymphoma patients andnormal monkeys treated with anti-B cell immunotoxins, B cell killingdefinitely occurs but causes no obvious harm (Vitetta et al., 1991).

D. ELAM-1

In contrast to Class II, ELAM-1 is not found on the vasculature ofnormal tissues in humans and is absent from any other cell types (Cotranet al., 1986). It is induced on vascular endothelial cells by IL-1 andTNV but not by IFN-γ (Wu et al., 1990). Its induction is rapid, peakingat 4-6 hours and, thereafter, it is rapidly lost, being hardlydetectable by 24 hours (Bevilacqua et al., 1987).

With ELAM-1, the strategy is to induce its expression selectively ontumor vasculature using a bispecific antibody that will home to thetumor and activate monocytes/macrophages within the tumor. Thebispecific antibody will have one Fab' arm directed against a tumorantigen and the other directed against CD14 (the LPS receptor). Afterlocalizing in the tumor, the bispecific antibody should crosslink CD14receptors on monocytes and the macrophages within the tumor. This shouldresult in powerful activation of these cells (Schutt et al., 1988; Chenet al., 1990) and the production of IL-1 and TNF which will induceELAM-1 on tumor vascular endothelial cells.

E. PREPARATION OF TARGETING AGENT ANTIBODIES

The origin or derivation of the targeting agent antibody or antibodyfragment (e.g., Fab', Fab or F(ab')₂) is not believed to be particularlycrucial to the practice of the invention, so long as the antibody orfragment that is actually employed for the preparation of bispecificantibodies otherwise exhibit the desired activating or bindingproperties. Thus, where monoclonal antibodies are employed, they may beof human, murine, monkey, rat, hamster, chicken or even rabbit origin.The invention contemplates that the use of human antibodies, "humanized"or chimeric antibodies from mouse, rat, or other species, bearing humanconstant and/or variable region domains, single domain antibodies (e.g.,DABs), Fv domains, as well as recombinant antibodies and fragmentsthereof. Of course, due to the ease of preparation and readyavailability of reagents, murine monoclonal antibodies will typically bepreferred.

In general, the preparation of bispecific antibodies is also well knownin the art, as exemplified by Glennie et al. (1987), as is their use inthe activation of leukocytes to release cytokines (Qian et al., 1991).Bispecific antibodies have even been employed clinically, for example,to treat cancer patients (Bauer et al., 1991). Generally speaking, inthe context of the present invention the most preferred method for theirpreparation involves the separate preparation of antibodies havingspecificity for the targeted tumor cell antigen, on the one hand, andthe targeted activating molecule on the other. While numerous methodsare known in the art for the preparation of bispecific antibodies, theGlennie et al. (1987) method preferred by the inventors involves thepreparation of peptic F(ab'γ)₂ fragments from the two chosen antibodies(e.g., an antitumor antibody and an anti-CD14 or anti-CD28 antibody),followed by reduction of each to provide separate Fab'γSH fragments. TheSH groups on one of the two partners to be coupled are then alkylatedwith a cross-linking reagent such as o-phenylenedimaleimide to providefree maleimide groups on one partner. This partner may then beconjugated to the other by means of a thioether linkage, to give thedesired F(ab'γ)₂ heteroconjugate.

While, due to ease of preparation and high yield and reproducibility,the Glennie et al. (1987) method is preferred for the preparation ofbispecific antibodies, there are of course numerous other approachesthat can be employed and that are envisioned by the inventors. Forexample, other techniques are known wherein crosslinking with SPDP orprotein A is carried out, or a trispecific construct is prepared (Tituset al., 1987; Tutt et al., 1991). Furthermore, recombinant technology isnow available for the preparation of antibodies in general, allowing thepreparation of recombinant antibody genes encoding an antibody havingthe desired dual specificity (Van Duk et al., 1989). Thus, afterselecting the monoclonal antibodies having the most preferred bindingand activation characteristics, the respective genes for theseantibodies can be isolated, e.g., by immunological screening of a phageexpression library (Oi & Morrison, 1986; Winter & Milstein, 1991). Then,through rearrangement of Fab coding domains, the appropriate chimericconstruct can be readily obtained.

The preparation of starting antibodies against the various cytokineactivating molecules is also well known in the art. For example, thepreparation and use of anti-CD14 and anti-CD28 monoclonal antibodieshaving the ability to induce cytokine production by leukocytes has nowbeen described by several laboratories (reviewed in Schutt et al., 1988;Chen et al., 1990, and June et al., 1990, respectively). Moreover, thepreparation of monoclonal antibodies that will stimulate leukocyterelease of cytokines through other mechanisms and other activatingantigens is also known (Clark et al., 1986; Geppert et al., 1990).

Similarly, there is a very broad array of antibodies known in the artthat have immunological specificity for the cell surface of virtuallyany solid tumor type, as a vast number of solid tumor assorted antigenshave been identified (see, e.g., Table II). Methods for the developmentof antibodies that are "custom-tailored" to the patient's tumor arelikewise known (Stevenson et al., 1990). Of course, not all antibodieswill have sufficient selectivity, specificity, affinity andtoxin-delivering capability to be of use in the practice of theinvention. These properties can be readily evaluated using conventionalimmunological screening methodology.

                                      TABLE II                                    __________________________________________________________________________    MARKER ANTIGENS OF SOLID TUMORS AND CORRESPONDING MONOCLONAL ANTIBODIES                  Antigen                                                                                               Identity/ Monoclonal                         Tumor Site Characteristics Antibodies Reference                             __________________________________________________________________________    A: Gynecological                                                                         `CA 125` >200                                                                             OC 125    Kabawat et al., Int. J. Gynecol. Pathol,       GY kD mucin GP  4:265, 1983; Szymendera, Tumour Biology,                         7:333, 1986                                                                ovarian 80 Kd GP OC 133 Masuko et al, Cancer Res., 44:2813, 1984                                              ovarian `SGA` 360 Kd GP OMI de Krester                                       et al., Int. J. Cancer, 37:705,                   1986                                                                       ovarian High M.sub.r mucin Mo v1 Miotti et al, Cancer Res., 65:826,                                          1985                                           ovarian High M.sub.r mucin/ Mo v2 Miotti et al, Cancer Res., 65:826,                                         1985                                            glycolipid                                                                   ovarian NS 3C2 Tsuji et al., Cancer Res., 45:2358, 1985                       ovarian NS 4C7 Tsuji et al., Cancer Res., 45:2358, 1985                       ovarian High M.sub.r mucin ID.sub.3 Gangopadhyay et al., Cancer Res.,                                        45:1744,                                          1985                                                                       ovarian High M.sub.r mucin DU-PAN-2 Lan et al, Cancer Res., 45:305,                                          1985                                           GY 7700 Kd GP F 36/22 Croghan etal., Cancer Res., 44:1954, 1984                                               ovarian `gp 68` 48 Kd 4F.sub.7 /7A.sub.1                                     0 Bhattacharya et al., Cancer Res.,                                           44:4528,                                        GP  1984                                                                     GY 40, 42kD GP OV-TL3 Poels et al., J. Natl. Cancer, 76:781, 1986                                             GY `TAG-72` High B72.3 Thor et al.,                                          Cancer Res., 46:3118, 1986                      M.sub.r mucin                                                                ovarian 300-400 Kd GP DF.sub.3 Kufe et al., Hybridoma, 3:223, 1984                                            ovarian 60 Kd GP 2C.sub.8 /2F.sub.7                                          Bhattacharya et al., Hybridoma, 4:153,                                        1985                                           GY 105 Kd GP MF 116 Mattes et al., PNAS, 81:568, 1984                         ovarian 38-40 kD GP MOv18 Miotti et al., Int. J. Cancer 39:297, 1987                                          GY `CEA` 180 Kd GP CEA 11-H5 Wagener et                                      al., Int. J. Cancer, 33:469, 1984                                              ovarian CA 19-9 or GICA CA 19-9 (1116NS                                      19- Atkinson et al., Cancer Res.,                                             62:6820, 1982                                    9)                                                                          ovarian `PLAP` 67 Kd GP H17-E2 McDicken et al., Br. J. Cancer, 52:59,                                        1985                                           ovarian 72 Kd 791T/36 Perkins et al., Eur. J. Nucl. Med., 10:296,                                                1985                                       ovarian 69 Kd PLAP NDOG.sub.2 Sunderland et al., Cancer Res., 44:4496,                                       1984                                           ovarian unknown M.sub.r PLAP H317 Johnson et al., Am. J. Reprod.                                             Immunol.,                                         1:246, 1981                                                                ovarian p185.sup.HER2 4D5, 3H4, 7C2, 6E9, Shepard et al., J. Clin.                                           Immunol., 11(3):117,                             2C4, 7F3, 2H11, 1991                                                          3E8, 5B8, 7D3, SB8                                                          uterus ovary HMFG-2 HMFG2 Epenetos et al., Lancet, Nov. 6, 1000-1004,                                            1982                                       GY HMFG-2 3.14.A3 Burchell et al., J. Immunol., 131:508, 1983                 B: BREAST 330-450 Kd GP DF3 Hayes et al., J. Clin. Invest., 75:1671,                                         1985                                            NS NCRC-11 Ellis et al., Histopathol., 8:501, 1984                            37kD 3C6F9 Mandeville et al., Cancer Detect. Prev.,                             10:89, 1987                                                                 NS MBE6 Teramoto et al., Cancer, 50:241, 1982                                 NS CLNH5 Glassy et al., PNAS, 80:63227, 1983                                  47 Kd GP MAC 40/43 Kjeldsen et al, 2nd Int. Wkshop of MAbs &                    Breast Cancer, San Fran., Nov. 1986                                         High M.sub.r GP EMA Sloane et al., Cancer, 17:1786, 1981                      High M.sub.r GP HMFG1 HFMG2 Arklie et al., Int. J. Cancer, 28:23, 1981        NS 3.15.C3 Arklie et al., Int. J. Cancer, 28:23, 1981                         NS M3, M8, M24 Foster et al., Virchows Arch. (Pathol. Anat.                     Histopathol.), 394:295, 1982                                                1 (Ma) blood M18 Foster et al., Humanpathol., 15:592, 1984                    group Ags                                                                     NS 67-D-11 Rasmussen et al., Breast Cancer Res. Treat.,                         2:401, 1982                                                                 oestrogen D547Sp, D75P3, H222 Kinsel et al., Cancer Res., 49:1052,                                          1989                                            receptor                                                                      EGF Receptor Anti-EGF Sainsbury et al, Lancet, 1:364, 1985                    Laminin LR-3 Horan Hand et al., Cancer Res., 45:2713, 1985.                   Receptor                                                                      erb B-2 p185 TA1 Gusterson et al., Br. J. Cancer, 58:453, 1988                                                NS H59 Hendler et al., Trans. Assoc.                                        Am. Physicians,                                   94:217, 1981                                                                126 Kd GP 10-3D-2 Soule et al., PNAS, 80:1332, 1983                           NS HmAB1, 2 Imam et al., cited in Schlom et al., Adv.                           Cancer Res., 43:143, 1985                                                   NS MBR 1, 2, 3 Menard et al., Cancer Res., 63:1295, 1983                      95 Kd 24 · 17 · 1 Thompson et al., J. Natl. Cancer                                        Inst.,                                            70:409, 1983                                                                100Kd 24 · 17 · 2 (3E1 · 2) Croghan et al.,                                      Cancer Res., 43:4980, 1983                      NS F36/22.M7/105 Croghan et al., Cancer Res., 44:1954, 1984                   24 Kd C11, G3, H7 Adams et al., Cancer Res., 43:6297, 1983                    90 Kd GP B6 · 2 Colcher et al., PNAS, 78:3199, 1981                  CEA & 180 Kd GP B1 · 1 Colcher et al., Cancer Invest., 1:127,                                      1983                                            colonic & Cam 17 · 1 Imperial Cancer Research Technology MAb                                         pancreatic  listing                           mucin similar                                                                 to Ca 19-9                                                                    milk mucin core SM3 Imperial Cancer Research Technology Mab                   protein  listing                                                              milk mucin core SM4 Imperial Cancer Research Technology Mab                   protein  listing                                                              affinity- C-Mul (566) Imperial Cancer Research Technology Mab                 purified milk  listing                                                        mucin                                                                         p185.sup.HER2 4D5 3H4, 7C2, 6E9, Shepard et al., J. Clin. Immunol.,                                         11(3) :117,                                      2C4, 7F3, 2H11, 1991                                                          3E8, 5B8, 7D3, 5B8                                                           CA 125 >200 Kd OC 125 Kabawat et al., Int. J. Gynecol. Pathol.,                                               GP  4:245, 1985                               High M.sub.r mucin/ MO v2 Miotti et al., Cancer Res., 45:826, 1985                                            glycolipid                                    High M.sub.r mucin DU-PAN-2 Lan et al., Cancer Res., 44 1954, 1984                                            `gp48` 48 Kd GP 4F.sub.7 /7A.sub.10                                         Bhattacharya et al , Cancer Res.,                                             44:4528,                                          1984                                                                        300-400 Kd GP DF.sub.3 Kufe et al., Hybridoma, 3:223, 1984                    `TAG-72` high B72 · 3 Thor et al., Cancer Res., 46:3118, 1986        M.sub.r mucin                                                                 `CEA` 180 Kd GP cccccCEA 11 Wagener et al., Int. J. Cancer, 33 469,                                         1984                                            `PLAP` 67 Kd GP H17-E2 McDicken et al., Br. J. Cancer, 52:59, 1985                                            HMFG-2 >400 Kd 3 · 14                                              · A3 Burchell et al., J.                                             Immunol., 131:508, 1983                         GP                                                                            NS FO23C5 Riva et al., Int. J. Cancer, 2:114, 1988                              (Suppl.)                                                                   C: COLORECTAL TAG-72 High M.sub.r B72 · 3 Colcher et al.,                                           Cancer Res., 47:1185 & 4218,                    mucin  1987                                                                   GP37 (17-1A) 1083-17-1A Paul et al., Hybridoma, 5:171, 1986                   Surface GP C017-1A LoBuglio et al., JNCl, 80:932, 1988                        CEA ZCE-025 Patt et al., Cancer Bull., 40:218, 1988                           CEA AB2 Griffin et al., Proc. 2nd Conf. on                                      Radioimmunodetection & Therapy of Cancer, 82,                                 1988                                                                        cell surface AG HT-29-15 Cohn et al., Arch. Surg. 122:1425, 1987                                              secretory 250-30.6 Leydem et al.,                                           Cancer, 57:1135, 1986                           epithelium                                                                    surface 44 × 14 Gallagher et al., J. Surg. Res., 40:159, 1986                                           glycoprotein                                  NS A7 Takahashi et al., Cancer, 61:881, 1988                                  NS GA73 · 3 Munz et al., J. Nucl, Med., 27:1739, 1986                                                NS 791T/36 Farrans et al., Lancet,                                          2:397, 1982                                     cell membrane & 28A32 Smith et al., Proc. Am. Soc. Clin. O. col.,                                             cytoplasmic Ag  6:250, 1987                   CEA & vindesine 28.19.8 Corvalen, Cancer Immuno., 24:133, 1987                                                gp72 X MMCO-791 Byers et al., 2nd Int.                                      Conf. Mab Immunocon.                              Cancer, 41:1987                                                             high M.sub.r mucin DU-PAN-2 Lan et al., Cancer Res., 45:305, 1985                                             high M.sub.r mucin ID.sub.3 Gangopadhya                                     y et al., Cancer Res., 45:1744,                   1985                                                                        CEA 180 Kd GP CEA 11-HS Wagener et al., Int. J. Cancer, 33:469, 1984                                          60 Kd GP 2C.sub.8 /2F.sub.7 Bhattachary                                     a et al., Hybridoma, 4:153, 1985                                                CA-19-9 (or CA-19-9 (1116NS 19-                                             Atkinson et al., Cancer Res., 62:6820,                                        1982                                            GICA) 9)                                                                      Lewis a PR5C5 Imperial Cancer Research Technology Mab                           Listing                                                                     Lewis a PR4D2 Imperial Cancer Research Technology Mab                           Listing                                                                     colonic mucus PR4D1 Imperial Cancer Research Technology Mab                     Listing                                                                    D. MELANOMA p97.sup.a 4 · 1 Woodbury et al., PNAS, 77:2183,                                         1980                                            p97.sup.a 8 · 2 M.sub.17 Brown, et al., PNAS, 78:539, 1981                                           p97.sup.b 96 · 5 Brown, et                                         al., PNAS, 78:539, 1981                         p97.sup.c 118 · 1, 133 · 2, Brown, et al., PNAS,                                          78:539, 1981                                     (113 · 2)                                                           p97.sup.c L.sub.1, L.sub.10, R.sub.10 (R.sub.19) Brown et al., J.                                           Immunol., 127:539, 1981                         p97.sup.d I.sub.12 Brown et al., J. Immunol., 127:539, 1981                   p97.sup.e K.sub.5 Brown et al., J. Immunol., 127:539, 1981                    p155 6 · 1 Loop et al., Int. J. Cancer, 27:775, 1981                 G.sub.D3 disialogan- R24 Dippold et al., PNAS, 77:6115, 1980                  glioside                                                                      p210, p60, p250 5.1 Loop et al., Int. J. Cancer, 27:775, 1981                 p280 p440 225.28S Wilson et al., Int. J. Cancer, 28:293, 1981                 GP 94, 75, 70 & 465.12S Wilson et al., Int. J. Cancer, 28:293, 1981                                           25                                            P240-P250, P450 9 · 2 · 27 Reisfeld et al., Melanoma                                      Ags & Abs, 1982 pp.                               317                                                                         100, 77, 75 Kd F11 Chee et al., Cancer Res., 42:3142, 1982                    94 Kd 376.96S Imai et al., JNCI, 68:761, 1982                                 4 GP chains 465.12S Imai et al., JNCI, 68:761, 1982; Wilson et                                                  al., Int. J. Cancer, 28:293, 1981                                           GP 74 15 · 75 Johnson &                                            Reithmuller, Hybridoma, 1:381, 1982                                             GP 49 15 · 95 Johnson &                                            Reithmuller, Hybridoma, 1:381, 1982                                             230 Kd Mel-14 Carrel et al., Hybridoma,                                      1:387, 1982                                    92 Kd Mel-12 Carrel et al., Hybridoma, 1:387, 1982                            70 Kd Me3-TB7 Carrel et al., Hybridoma, 1:387, 1982                           HMW MAA similar 225.28SD Kantor et al., Hybridoma, 1:473, 1982                                                to 9 · 2 · 27 AG                                            HMW MAA similar 763.24TS Kantor et                                          al., Hybridoma, 1:473, 1982                     to 9 · 2 · 27 AG                                            GP95 similar to 705F6 Stuhlmiller et al., Hybridoma, 1:447, 1982                                              376 · 96S 465 · 12S                                         GP125 436910 Saxton et al., Hybridoma,                                      1:433, 1982                                     CD41 M148 Imperial Cancer Research Technology Mab                               listing                                                                    E: high M.sub.r mucin ID3 Gangopadhyay et al., Cancer Res., 45:1744,                                          GASTROINTESTINAL   1985                       pancreas, stomach                                                             gall bladder, high M.sub.r mucin DU-PAN-2 Lan et al., Cancer Res.,                                           45:305, 1985                                   pancreas, stomach                                                             pancreas NS OV-TL3 Poels et al., J. Natl. Cancer Res., 44:4528,                                                  1984.                                      pancreas, stomach, `TAG-72` high B72 · 3 Thor et al., Cancer                                        Res., 46:3118, 1986                            oesophagus M.sub.r mucin                                                      stomach `CEA` 180 Kd GP CEA 11-H5 Wagener et al., Int. J. Cancer,                                            33:469, 1984                                   pancreas HMFG-2 >400 Kd 3 · 14 · A3 Burchell et al.,                                       J. Immunol., 131:508, 1983                      GP                                                                           G · I · NS C COLI Lemkin et al., Proc. Am. Soc. Clin.                                      Oncol.,                                           3:47, 1984                                                                 pancreas, stomach CA 19-9 (or CA-19-9 (1116NS 19- Szymendera, Tumour                                         Biology, 7:333, 1986                            GICA) 9) and CA50                                                            pancreas CA125 GP OC125 Szymendera, Tumour Biology, 7:333, 1986                                               F: LUNG p185.sup.HER2 4D5 3H4, 7C2,                                          6E9, Shepard et al., J. Clin. Immunol.,                                       11(3):117,                                     non-small cell  2C4, 7F3, 2H11, 1991                                          lung carcinoma  3E8, 5B8, 7D3, SB8                                             high M.sub.r mucin/ MO v2 Miolti et al., Cancer Res., 65:826, 1985                                            glycolipid                                    `TAG-72` high B72 · 3 Thor et al., Cancer Res., 46:3118, 1986        M.sub.r mucin                                                                 high M.sub.r mucin DU-PAN-2 Lan et al., Cancer Res., 45:305, 1985                                             `CEA` 180 kD GP CEA 11-H5 Wagener et                                        al, Int. J. Cancer., 33:469, 1984                                              Malignant Gliomas cytoplasmic MUC 8-22                                       Stavrou, Neurosurg. Rev., 13:7, 1990                                            antigen from                                  85HG-22 cells                                                                 cell surface Ag MUC 2-63 Stavrou, Neurosurg. Rev., 13:7, 1990                 from 85HG-63                                                                  cells                                                                         cell surface Ag MUC 2-39 Stavrou, Neurosurg. Rev., 13:7, 1990                 from 86HG-39                                                                  cells                                                                         cell surface Ag MUC 7-39 Stavrou, Neurosurg. Rev., 13:7, 1990                 from 86HG-39                                                                  cells                                                                        G: MISCELLANEOUS p53 PAb 240 Imperial Cancer Research Technology MaB                                            PAb 246 Listing                               PAb 1801                                                                    small round cell neural cell ERIC · 1 Imperial Cancer Research                                      Technology MaB                                 tumors adhesion  Listing                                                       molecule                                                                     medulloblastoma  M148 Imperial Cancer Research Technology MaB                 neuroblastoma   Listing                                                       rhabdomyosarcoma                                                              neuroblastoma  FMH25 Imperial Cancer Research Technology MaB                     Listing                                                                    renal cancer & p155 6 · 1 Loop et al., Int. J. Cancer, 27:775,                                      1981                                           glioblastomas                                                                 bladder & "Ca Antigen" CA1 Ashall et al., Lancet, July 3, 1, 1982                                             laryngeal cancers 350-390 kD                  neuroblastoma GD2 3F8 Cheung et al., Proc. AACR, 27:318, 1986                 Prostate gp48 48 kD GP 4F.sub.7 /7A.sub.10 Bhattacharya et al., Cancer                                       Res. 44:4528,                                     1984                                                                       Prostate 60 kD GP 2C.sub.8 /2F.sub.7 Bhattacharya et al., Hybridoma,                                         4:153, 1985                                    Thyroid `CEA` 180 kD GP CEA 11-H5 Wagener et al., Int. J. Cancer,                                            33:469, 1984                                 __________________________________________________________________________     abbreviations: Abs, antibodies; Ags, antigens; EGF, epidermal growth          factor; GI, gastrointestinal; GICA, gastrointestinalassociated antigen;       GP, glycoprotein; GY, gynecological; HMFG, human milk fat globule; Kd,        kilodaltons; Mabs, monoclonal antibodies; M.sub.r, molecular weight; NS,      not specified; PLAP, placental alkaline phosphatase; tag, tumorassociated     glycoprotein; CEA, carcinoembryonic antigen.                                  footnotes:                                                                    the CA 19-9 Ag (GICA) is sialosylfucosyllactotetraosylceramide, also          termed sialylated Lewis pentaglycosyl ceramide or siarylated                  lactoN-fucopentaose II; p97 Ags are believed to be chondroitin sulphate       proteoglycan; antigens reactive with Mab 9 · 2 · 27 are     believed to be sialylated glycoproteins associated with chondroitin           sulphate proteoglycan; unless specified, GY can include cancers of the        cervix, endocervix, endometrium, fallopian tube, ovary, vagina or mixed       Mullerian tumor; unless specified GI can include cancers of the liver,        small intestine, spleen, pancreas, stomach and oesophagus.               

Generally speaking, antibodies of the present invention will preferablyexhibit properties of high affinity, such as exhibiting a K_(d) of <200nM, and preferably, of <100 nM, and will not show significant reactivitywith life-sustaining normal tissues, such as one or more tissuesselected from heart, kidney, brain, liver, bone marrow, colon, breast,prostate, thyroid, gall bladder, lung, adrenals, muscle, nerve fibers,pancreas, skin, or other life-sustaining organ or tissue in the humanbody. The "life-sustaining" tissues that are the most important for thepurposes of the present invention, from the standpoint of lowreactivity, include heart, kidney, central and peripheral nervous systemtissues and liver. The term "significant reactivity", as used herein,refers to an antibody or antibody fragment, which, when applied to theparticular tissue under conditions suitable for immunohistochemistry,will elicit either no staining or negligible staining with only a fewpositive cells scattered among a field of mostly negative cells. Many ofthe antibodies listed in Table II will not be of sufficient tumorspecificity to be of use therapeutically. An example is MUC8-22 whichrecognizes a cytoplasmic antigen. Antibodies such as these will be ofuse only in diagnostic or investigational embodiments such as in modelsystems or screening assays.

Particularly promising antibodies from the stand point of therapeuticapplication of the present invention are those having high selectivityfor the solid tumor, such as B72.3, PR5C5 or PR4D2 for colorectaltumors; HMFG-2, TAG 72, SM-3, or anti-p 185^(Her2) for breast tumors;anti-p 185^(Her2) for lung tumors; 9.2.27 for melanomas; and MO v18 andOV-TL3 for ovarian tumors.

The listing of potential solid tumor cell surface antigen targets inTable II is intended to be illustrative rather than exhaustive. Ofcourse, in the practice of the invention, one will prefer to ensure inadvance that the clinically-targeted tumor expresses the antigenultimately selected. This is a fairly straightforward assay, involvingantigenically testing a tumor tissue sample, for example, a surgicalbiopsy, or perhaps testing for circulating shed antigen. This canreadily be carried out in an immunological screening assay such as anELISA (enzyme-linked immunosorbent assay), wherein the binding affinityof antibodies from a "bank" of hybridomas are tested for reactivityagainst the tumor. Antibodies demonstrating appropriate tumorselectivity and affinity are then selected for the preparation ofbispecific antibodies of the present invention. Antitumor antibodieswill also be useful in the preparation of antitumor antibody conjugatesfor use in combination regimens, wherein tumor endothelium and the solidtumor itself are both targeted (see, e.g., FIG. 12).

F. PREPARATION OF TARGETING AGENT/TOXIN COMPOUNDS; INCLUDINGIMMUNOTOXINS

Methods for the production of the target agent/toxin agent compounds ofthe invention are described herein. The targeting agents, such asantibodies, of the invention may be linked, or operatively attached, tothe toxins of the invention by either crosslinking or via recombinantDNA techniques, to produce, for example, targeted immunotoxins.

While the preparation of immunotoxins is, in general, well known in theart (see, e.g., U.S. Pat. No. 4,340,535, and EP 44167, both incorporatedherein by reference), the inventors are aware that certain advantagesmay be achieved through the application of certain preferred technology,both in the preparation of the immunotoxins and in their purificationfor subsequent clinical administration. For example, while IgG basedimmunotoxins will typically exhibit better binding capability and slowerblood clearance than their Fab' counterparts, Fab' fragment-basedimmunotoxins will generally exhibit better tissue penetrating capabilityas compared to IgG based immunotoxins.

Additionally, while numerous types of disulfide-bond containing linkersare known which can successfully be employed to conjugate the toxinmoiety with the targeting agent, certain linkers will generally bepreferred over other linkers, based on differing pharmacologiccharacteristics and capabilities. For example, linkers that contain adisulfide bond that is sterically "hindered" are to be preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action. Furthermore, whilecertain advantages in accordance with the invention will be realizedthrough the use of any of a number of toxin moieties, the inventors havefound that the use of ricin A chain, and even more preferablydeglycosylated A chain, will provide particular benefits.

A wide variety of cytotoxic agents are known that may be conjugated toanti-endothelial cell antibodies. Examples include numerous usefulplant-, fungus- or even bacteria-derived toxins, which, by way ofexample, include various A chain toxins, particularly ricin A chain,ribosome inactivating proteins such as saporin or gelonin, α-sarcin,aspergillin, restrictocin, ribonucleases such as placental ribonuclease,angiogenic, diphtheria toxin, and pseudomonas exotoxin, to name just afew. The most preferred toxin moiety for use in connection with theinvention is toxin A chain which has been treated to modify or removecarbohydrate residues, so called deglycosylated A chain. The inventorshave had the best success through the use of deglycosylated ricin Achain (dgA) which is now available commercially from InlandLaboratories, Austin, Tex.

However, it may be desirable from a pharmacologic standpoint to employthe smallest molecule possible that nevertheless provides an appropriatebiological response. One may thus desire to employ smaller A chainpeptides which will provide an adequate anti-cellular response. To thisend, it has been discovered by others that ricin A chain may be"truncated" by the removal of 30 N-terminal amino acids by Nagarase(Sigma), and still retain an adequate toxin activity. It is proposedthat where desired, this truncated A chain may be employed in conjugatesin accordance with the invention.

Alternatively, one may find that the application of recombinant DNAtechnology to the toxin A chain moiety will provide additionalsignificant benefits in accordance the invention. In that the cloningand expression of biologically active ricin A chain has now been enabledthrough the publications of others (O'Hare et al., 1987; Lamb et al.,1985; Halling et al., 1985), it is now possible to identify and preparesmaller or otherwise variant peptides which nevertheless exhibit anappropriate toxin activity. Moreover, the fact that ricin A chain hasnow been cloned allows the application of site-directed mutagenesis,through which one can readily prepare and screen for A chain derivedpeptides and obtain additional useful moieties for use in connectionwith the present invention.

The cross-linking of the toxin A chain region of the conjugate with thetargeting agent region is an important aspect of the invention. Incertain cases, it is required that a cross-linker which presentsdisulfide function be utilized for the conjugate to have biologicalactivity. The reason for this is unclear, but is likely due to a needfor certain toxin moieties to be readily releasable from the targetingagent once the agent has "delivered" the toxin to the targeted cells.Each type of cross-linker, as well as how the cross-linking isperformed, will o tend to vary the pharmacodynamics of the resultantconjugate. Ultimately, in cases where a releasable toxin iscontemplated, one desires to have a conjugate that will remain intactunder conditions found everywhere in the body except the intended siteof action, at which point it is desirable that the conjugate have good"release" characteristics. Therefore, the particular cross-linkingscheme, including in particular the particular cross-linking reagentused and the structures that are cross-linked, will be of somesignificance.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different proteins (e.g., a toxin anda binding agent). To link two different proteins in a step-wise manner,heterobifunctional cross-linkers can be used which eliminate theunwanted homopolymer formation. An exemplary heterobifunctionalcross-linker contains two reactive groups: one reacting with primaryamine group (e.g., N-hydroxy succinimide) and the other reacting with athiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).Through the primary amine reactive group, the cross-linker may reactwith the lysine residue(s) of one protein (e.g., the selected antibodyor fragment) and through the thiol reactive group, the cross-linker,already tied up to the first protein, reacts with the cysteine residue(free sulfhydryl group) of the other protein (e.g., dgA).

The spacer arm between these two reactive groups of any cross-linkersmay have various length and chemical composition. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents).

The most preferred cross-linking reagent is SMPT, which is abifunctional cross-linker containing a disulfide bond that is"sterically hindered" by an adjacent benzene ring and methyl groups. Itis believed that stearic hindrance of the disulfide bond serves afunction of protecting the bond from attack by thiolate anions such asglutathione which can be present in tissues and blood, and thereby helpin preventing decoupling of the conjugate prior to its delivery to thesite of action by the binding agent. The SMPT cross-linking reagent, aswith many other known cross-linking reagents, lends the ability tocross-link functional groups such as the SH of cysteine or primaryamines (e.g., the epsilon amino group of lysine). Another possible typeof cross-linker includes the heterobifunctional photoreactivephenylazides containing a cleavable disulfide bond such assulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3'-dithiopropionate.The N-hydroxy-succinimidyl group reacts with primary amino groups andthe phenylazide (upon photolysis) reacts non-selectively with any aminoacid residue.

Although the "hindered" cross-linkers will generally be preferred in thepractice of the invention, non-hindered linkers can be employed andadvantages in accordance herewith nevertheless realized. Other usefulcross-linkers, not considered to contain or generate a protecteddisulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe,1987). The use of such cross-linkers is well understood in the art.

Once conjugated, it will be important to purify the conjugate so as toremove contaminants such as unconjugated toxin or targeting agent. It isimportant to remove unconjugated A chain because of the possibility ofincreased toxicity. Moreover, it is important to remove unconjugatedtargeting agent to avoid the possibility of competition for the antigenbetween conjugated and unconjugated species. In any event, a number ofpurification techniques are disclosed in the Examples below which havebeen found to provide conjugates to a sufficient degree of purity torender them clinically useful.

In general, the most preferred technique will incorporate the use ofBlue-Sepharose with a gel filtration or gel permeation step.Blue-Sepharose is a column matrix composed of Cibacron Blue 3GA andagarose, which has been found to be useful in the purification ofimmunoconjugates (Knowles & Thorpe, 1987). The use of Blue-Sepharosecombines the properties of ion exchange with A chain binding to providegood separation of conjugated from unconjugated binding.

The Blue-Sepharose allows the elimination of the free (non conjugated)targeting agent (e.g., the antibody or fragment) from the conjugatepreparation. To eliminate the free (unconjugated) toxin (e.g., dgA) amolecular exclusion chromatography step is preferred using eitherconventional gel filtration procedure or high performance liquidchromatography. One may also use the methods disclosed in U.S. patentapplication Ser. No. 08/147,768, incorporated herein by reference, whichenables the production of immunotoxins bearing one, two or three toxinchains per antibody molecule.

Standard recombinant DNA techniques that are well known to those ofskill in the art may be utilized to express nucleic acids encoding thetargeting agent/toxin compounds of the invention. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. DNA and RNA synthesismay, additionally, be performed using an automated synthesizers (see,for example, the techniques described in Sambrook et al., 1989; andAusubel et al., 1989).

When produced via recombinant DNA techniques such as those describedherein, the targeting agent/toxin compounds of the invention may bereferred to herein as "fusion proteins". It is to be understood thatsuch fusion proteins contain at least a targeting agent and a toxin ofthe invention, operatively attached. The fusion proteins may alsoinclude additional peptide sequences, such as peptide spacers whichoperatively attach the targeting agent and toxin compound, as long assuch additional sequences do not appreciably affect the targeting ortoxin activities of the fusion protein.

Depending on the specific toxin compound used as part of the fusionprotein, it may be necessary to provide a peptide spacer operativelyattaching the targeting agent and the toxin compound which is capable offolding into a disulfide-bonded loop structure. Proteolytic cleavagewithin the loop would then yield a heterodimeric polypeptide wherein thetargeting agent and the toxin compound are linked by only a singledisulfide bond. See, for example, Lord et al. (1992). An example of sucha toxin is a Ricin A-chain toxin.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the targeting agent and thetoxin compound of the fusion protein. Toxins which may be used inconjunction with non-cleavable peptide spacers are those which may,themselves, be converted by proteolytic cleavage, into a cytotoxicdisulfide-bonded form (see for example, Ogata et al., 1990). An exampleof such a toxin compound is a Pseudonomas exotoxin compound.

Nucleic acids that may be utilized herein comprise nucleic acidsequences that encode a targeting agent of interest and nucleic acidsequences that encode a toxin agent of interest. Such targetagent-encoding and toxin agent-encoding nucleic acid sequences areattached in a manner such that translation of the nucleic acid yieldsthe targeting agent/toxin compounds of the invention.

Standard techniques, such as those described above may be used toconstruct expression vectors containing the above-described nucleicacids and appropriate transcriptional/translational control sequences. Avariety of host-expression vector systems may be utilized. These includebut are not limited to microorganisms such as bacteria (e.g., E. coli,B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNAor cosmid DNA expression vectors containing targeting agent/toxin codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing targeting agent/toxincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the targetingagent/toxin coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing the targetingagent/toxin coding sequences coding sequence; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for thetargeting agent/toxin compound being expressed. For example, when largequantities of targeting agent/toxin compound are to be produced for thegeneration of antibodies or to screen peptide libraries, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include but are notlimited to the E. coli expression vector pUR278 (Ruther et al., 1983),in which the targeting agent/toxin coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein additionally containing a portion of the lac Zproduct is provided; pIN vectors (Inouye et al., 1985; Van Heeke et al.,1989); and the like. pGEX vectors may also be used to express foreignpolypeptides, such as the targeting agent/toxin compounds as fusionproteins additionally containing glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the targeting agent/toxin protein of the fusion protein can bereleased from the GST moiety.

In an insect system, Autograph californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The targeting agent/toxin coding sequencesmay be cloned into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of the targetingagent/toxin coding sequences will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed (e.g., see Smith et al.,1983; Smith U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the targeting agent/toxin coding sequences may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing targeting agent/toxin proteins in infected hosts (e.g., seeLogal et al., 1984). Specific initiation signals may also be requiredfor efficient translation of inserted targeting agent/toxin codingsequences. These signals include the ATG initiation codon and adjacentsequences. Exogenous translational control signals, including the ATGinitiation codon, may additionally need to be provided. One of ordinaryskill in the art would readily be capable of determining this andproviding the necessary signals. Furthermore, the initiation codon mustbe in phase with the reading frame of the desired coding sequence toensure translation of the entire insert. These exogenous translationalcontrol signals and initiation codons can be of a variety of origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. Forlong-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressconstructs encoding the targeting agent/toxin compounds may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with targetingagent/toxin DNA controlled by appropriate expression control elements(e.g., promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of foreign DNA, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limited,to the herpes simplex virus thymidine kinase (Wigler et al., 1977),hypoxanthine-guanine phosphoriboxyltransferase (Szybalska et al., 1962),and adenine phosphoriboxyltransferase genes (Lowy et al., 1980) can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980;O'Hare et al., 1981); gpt, which confers resistance to mycophenolic acid(Mulligan et al., 1981); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984).

After a sufficiently purified compound has been prepared, one willdesire to prepare it into a pharmaceutical composition that may beadministered parenterally. This is done by using for the lastpurification step a medium with a suitable pharmaceutical composition.

Suitable pharmaceutical compositions in accordance with the inventionwill generally comprise from about 10 to about 100 mg of the desiredconjugate admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a finalconcentration of about 0.25 to about 2.5 mg/ml with respect to theconjugate. Such formulations will typically include buffers such asphosphate buffered saline (PBS), or additional additives such aspharmaceutical excipients, stabilizing agents such as BSA or HSA, orsalts such as sodium chloride. For parenteral administration it isgenerally desirable to further render such compositions pharmaceuticallyacceptable by insuring their sterility, non-immunogenicity andnon-pyrogenicity. Such techniques are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. It should beappreciated that endotoxin contamination should be kept minimally at asafe level, for example, less that 0.5 ng/mg protein. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

A preferred parenteral formulation of the targeting agent/toxincompounds, including immunotoxins, in accordance with the presentinvention is 0.25 to 2.5 mg conjugate/ml in 0.15M NaCl aqueous solutionat pH 7.5 to 9.0. The preparations may be stored frozen at -10° C. to-70° C. for at least 1 year.

G. ATTACHMENT OF OTHER AGENTS TO TARGETING AGENTS

It is contemplated that most therapeutic applications of the presentinvention will involve the targeting of a toxin moiety to the tumorendothelium. This is due to the much greater ability of most toxins todeliver a cell killing effect as compared to other potential agents.However, there may be circumstances, such as when the target antigendoes not internalize by a route consistent with efficient intoxicationby targeting agent/toxin compounds, such as immunotoxins, where one willdesire to target chemotherapeutic agents such as antitumor drugs, othercytokines, antimetabolites, alkylating agents, hormones, and the like.The advantages of these agents over their non-targeting agent conjugatedcounterparts is the added selectivity afforded by the targeting agent,such as an antibody. One might mention by way of example agents such assteroids, cytosine arabinoside, methotrexate, aminopterin,anthracyclines, mitomycin C, vinca alkaloids, demecolcine, etoposide,mithramycin, and the like. This list is, of course, merely exemplary inthat the technology for attaching pharmaceutical agents to targetingagents, such as antibodies, for specific delivery to tissues is wellestablished (see, e.g., Ghose & Blair, 1987).

It is proposed that particular benefits may be achieved through theapplication of the invention to tumor imaging. Imaging of the tumorvasculature is believed to provide a major advantage when compared topresent imaging techniques, in that the cells are readily accessible.Moreover, the technology for attaching paramagnetic, radioactive andeven fluorogenic ions to targeting agents, such as antibodies, is wellestablished. Many of these methods involve the use of a metal chelatecomplex employing, for example, an organic chelating agent such a DTPAattached to the antibody (see, e.g., U.S. Pat. No. 4,472,509). In thecontext of the present invention the selected ion is thus targeted tothe tumor endothelium by the targeting agent, such as an antibody,allowing imaging to proceed by means of the attached ion.

A variety of chemotherapeutic and other pharmacologic agents have nowbeen successfully conjugated to antibodies and shown to functionpharmacologically (see, e.g., Vaickus et al., 1991). Exemplaryantineoplastic agents that have been investigated include doxorubicin,daunomycin, methotrexate, vinblastine, and various others (Dillman etal., 1988; Pietersz et al., 1988). Moreover, the attachment of otheragents such as neocarzinostatin (Kimura et al., 1983), macromycin(Manabe et al., 1984), trenimon (Ghose, 1982) and α-amanitin (Davis &Preston, 1981) has been described.

In addition to chemotherapeutic agents, the inventors contemplate thatthe invention will be applicable to the specific delivery of a widevariety of other agents to tumor vasculature. For example, under certaincircumstances, one may desire to deliver a coagulant such as Russell'sViper Venom, activated Factor IX, activated Factor X or thrombin to thetumor vasculature. This will result in coagulation of the tumor's bloodsupply. One can also envisage targeting a cell surface lytic agent suchas phospholipase C (Flickinger & Trost, 1976) or cobra venom factor(CVF) (Vogel & Muller-Eberhard, 1981) which should lyse the tumorendothelial cells directly. The operative attachment of such structuresto targeting agents, such as antibodies, may be readily accomplished,for example, by protein- protein coupling agents such as SMPT. Moreover,one may desire to target growth factors, other cytokines or evenbacterial endotoxin or the lipid A moiety of bacterial endotoxin to aselected cell type, in order, e.g., to achieve modulation of cytokinerelease. The attachment of such substances is again well within theskill in the art as exemplified by Ghose & Blair (1987).

Thus, it is generally believed to be possible to conjugate to antibodiesany pharmacologic agent that has a primary or secondary amine group,hydrazide or hydrazine group, carboxyl alcohol, phosphate, or alkylatinggroup available for binding or cross-linking to the amino acids orcarbohydrate groups of the antibody. In the case of protein structures,this is most readily achieved by means of a cross linking agent (seepreceding section on immunotoxins). In the case of doxorubicin anddaunomycin, attachment may be achieved by means of an acid labile acylhydrazone or cis aconityl linkage between the drug and the antibody.Finally, in the case of methotrexate or aminopterin, attachment isachieved through a peptide spacer such as L-Leu-L-Ala-L-Leu-L-Ala,between the γ-carboxyl group of the drug and an amino acid of theantibody. For a general overview of linking technology, one may wish torefer to Ghose & Blair (1987).

Alternatively, any such structures which are nucleic acid-encodedstructures may be operatively attached to the targeting agents of theinvention by standard recombinant DNA techniques, such as, for example,those discussed above, in the previous section.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLE I A Murine Model for Antibody-Directed Targeting of VascularEndothelial Cells in Solid Tumors

This example describes the development of a model system in which toinvestigate the antibody-directed targeting of vascular endothelialcells in solid tumors in mice. A neuroblastoma transfected with themouse interferon-γ (IFN-γ) gene, C1300(Muγ), was grown in SCID andantibiotic-treated BALB/c nude mice. The INF-γ secreted by the tumorinduces the expression of MHC Class II antigens on the tumor vascularendothelium. Class II antigens are absent from the vasculature of normaltissues, although they are present on B-lymphocytes, cells ofmonocyte/macrophage lineage and some epithelial cells.Intravenously-administered anti-Class II antibody strongly stains thetumor vasculature whereas an anti-tumor antibody, directed against a MHCClass I antigen of the tumor allograft, produces classical perivasculartumor cell staining.

A. Materials and Methods

1. Animals

BALB/c nu/nu mice were purchased from Simonsen (Gilroy, Calif.). SCIDmice were from the UT Southwestern Medical Center breeding colony. Allanimals were maintained in microisolation units on sterilized food andwater. Where indicated, tetracycline--HCI (Vedeo, St. Joseph, Mo.) wasadded to drinking water to a final concentration of 1.1 mg/ml (Harknesset al., 1983). Both strains carry the H-2^(d) haplotype.

2. Cells and Culture Conditions

All cell lines used in this study were cultured in modified Eagle'smedium (MEM) supplemented with 10 (v/v) fetal calf serum, 2.4 mML-glutamine, 200 units/ml penicillin and 100 μg/ml streptomycin.Cultures were maintained at 37° C. in a humidified atmosphere of 90%air/10 CO₂. The C1300 neuroblastoma line was established from aspontaneous tumor which arose in an A/Jax mouse in 1940 (Dunham et al.,1953). The C1300(Muγ)12 line, hereafter abbreviated to C1300 (Muγ), wasderived by transfection of C1300 cells with murine IFN-γ gene using theIFN-γ expression retrovirus pSVX (MuγδA_(s)) (Watanabe et al., 1988;Watanabe et al., 1989), and was cultured in MEM as above containing 1mg/ml G418 (Geneticin, Sigma). Both lines carry the MHC haplotypeH-2K^(k), I-A^(k), I-E^(k), D^(d). C1300 and C1300(Muγ) cells were grownin regular tissue culture flasks or, when large quantities were requiredfor in vivo studies, in cell factories (Baxter, Grand Prairie, Tex.).Cells from subcutaneous tumors were recovered for in vitro analysis bygentle mincing in MEM. After tumor cells had adhered overnight themonolayers were washed twice with MEM to remove nonadherent contaminanthost cells.

Tumor conditioned media were prepared by seeding C1300 and C1300(Muγ)cells at 25 of confluent density and culturing them for four days.Conditioned media were dialyzed for 16 hours against MEM without FCS toremove G418, filtered through a 0.22 μM membrane and stored at 4° C. forno more than one week before assay. Aliquots of anti-IFN-γ antibodies(see `Monoclonal Antibodies`) sufficient to neutralize 200 internationalunits (I.U.) of murine IFN-γ/ml of conditioned medium were added to somesamples 24 hours before assay. The SVEC-10 murine endothelial cell line,hereafter abbreviated to SVEC, was kindly provided to Dr. M. Edidin,Department of Biology, Johns Hopkins University, Baltimore, MD and wasderived by immortalization of lymph node endothelial cells from a C3H(H-2^(k)) mouse with SV40 (O'Connell et al., 1990). For some studies,SVEC cells were cultured for 72 hours with 100 I.U./ml recombinantmurine IFN-γ, (r.IFN-γ, a generous gift from Dr. F. Balkwill, ImperialCancer Research Fund, London, England) or tumor-conditioned medium. Inaddition, 200 I.U./ml anti-IFN-γ antibody was added to some flasks atthe beginning of the 72 hour culture period.

3. Monoclonal Antibodies

The M5/114.15.2 (hereafter abbreviated to M5/114) and 11-4.1 hybridomaswere purchased from the American Type Collection (Rockville, Md.) andwere grown in MEM-10% FCS. The antibodies were purified from culturesupernatant by precipitation in 50% ammonium sulphate and affinitychromatography on Protein A. The rat IgG2b antibody, M5/114, detects anIa specificity on I-A^(b), I-A^(q), IA^(d), I-E^(d) and I-E^(k)molecules (Bhattacharya et al., 1981). Thus, the antibody recognizesI-E^(k) molecules on SVEC (H-2^(k)) cells and I-A^(d) and I-E^(d),hereafter referred to collectively as Ia^(d), on cells from BALB/C nu/nuor SCID mice (both H-2^(d)). The anti-Ia^(d) reactivity of M5/114 wasconfirmed in this study by FACS analyses with the Ia^(d) expressingB-lymphoma line, A20/25 (Kim, 1978). The mouse IgG2a antibody 11-4.1recognizes H-2K^(k) but not H-2K^(d) molecules (Oi et al., 1978) and sobinds to H-2K^(k) on C1300 and C1300(Muγ) cells but is unreactive withMHC antigens from BALB/c nu/nu or SCID mice. Isotype-matched controlantibodies of irrelevant specificity were CAMPATH-2 (rat IgG2b,anti-human CD7 (Bindon et al., 1988) and WT-1 (mouse IgG2a, anti-humanCD7 (Tax et al., 1984). Purified preparations of CAMPATH-2 and WT-1 weregenerous gifts from Dr. G. Hale (Department of Pathology, Cambridge,England) and Dr. W. Tax (Sint Radboudzeikenhuis, Nijmegen, theNetherlands) respectively.

Rat anti-mouse endothelial cell antibody MECA-20 (Duijvestijn et al.,1987) was donated as a concentrated culture supernatant by Dr. A.Duijvestijn (University of Limburg, the Netherlands) and used at adilution of 1/200 for indirect immunoperoxidase staining. Rat antibodiesagainst mouse macrophages (Ml) and mouse CD3 (KT 31.1) were generouslyprovided by Dr. P. Beverley (Imperial Cancer Research Fund, London,England). Hamster anti-mouse IFN-γ antibody 1222-00 (Sanchez-Madrid,1983), used for specific neutralization of IFN-γ in vitro, was purchasedfrom Genzyme (Boston, Mass.). Anti-mouse IFN-γ antibodies, XMG1.2 andR46A2, used in IFN-γ ELISAs, were kindly provided by Dr. N. Street (U.T.Southwestern Medical Center, Dallas, Tex.). Purified 11-4.1, WT-1 andXMG1.2 antibodies were biotinylated by incubation with a 12.5 fold molarexcess of N-hydroxysuccinimidobiotin amidocaproate (Sigma) for one hourat room temperature followed by dialysis against two changes of PBS.

4. ELISA for murine IFN-γ

Sandwich ELISAs for murine IFN-γ were carried out as described by(Cherwinski et al., 1989). The wells of flexible PVC microtiter plates(Dynatech, Alexandria, Va.) were coated with 50 μl/well of a 2 μg/mlsolution of capture anti-IFN-γ antibody, R46A2, in PBS for 2 hours atroom temperature. Non-specific protein binding sites were blocked with20% FCS in PBS for 15 minutes at 37° C. The plates were washed threetimes in PBS containing 0.05% (v/v) Tween 20 (Sigma) (PBS-T) and 25μl/well control and test samples in MEM-10% FCS were added. Afterincubating for 1 hour at 37° C. , the wells were washed as before and 50μl/well of a 1 μg/ml solution of biotinylated anti-IFN-γ antibody XMG1.2in PBS-T containing 1% BSA were added. After incubation for 30 minutesat 37° C. the wells were washed as before and incubated with 75 μl of a1:2000 dilution of horseradish peroxidase-conjugated streptavidin (DAKO)for one hour at room temperature. After thorough washing in PBS-T thewells were incubated for 30 minutes with 100 μl/well of a 1 mg/mlsolution of 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS, Sigma) in citrate/phosphate butter containing 0.003% (v/v) H₂ O₂.Reaction product was measured as Abs.405 nm--Abs.490 nm. IFN-γ levels intest samples were calculated by reference to a recombinant murine IFN-γstandard solution in MEM-10% FCS.

5. Indirect Immunofluorescence

SVEC, C1300 and C1300(Muγ) cells were prepared for FACS analyses asdescribed by Burrows et al. (1991). All manipulations were carried outat room temperature. 50 μl of a cell suspension at 2-3×10⁶ cells/ml inPBS containing 0.2% (w/v) BSA and 0.2% (w/v) NaN₃ (PBS-BSA-N₃) wereadded to the wells of round-bottomed 96-well microtiter plates (Falcon3910). Optimal dilutions of rat or mouse antibodies were distributed in50 μl volumes, and the plates sealed. After 15 minutes, the cells werewashed four times by centrifuging the plates at 800×g for 30 seconds,removing the supernatants, and resuspending the cells in 150 μl/wellPBS-BSA-N3. Fluorescein isothiocyanate-conjugated rabbit antibodiesagainst rat or mouse IgG (ICN, High Wycombe, England), diluted 1:20 inPBS-BSA-N₃ were distributed in 50 μl volumes into the appropriate wells.The cells were incubated for a further 15 minutes and washed as before.Cell-associated fluorescence was measured on a FACScan (BectonDickenson, Fullerton, Calif.). Data were analyzed using the CONSORT 30program.

6. Preparation of tissues and immunohistochemistry For the establishmentof solid tumors, a total of 2×10⁷ C1300 or C1300(Muγ) cells, or amixture of the two, in 200 μl MEM-30% FCS were injected subcutaneouslyinto the right anterior flank of BALB/c nu/nu or SCID mice. Tumordiameters were measured at regular intervals and the animals wereeuthanized after 16 days (rapidly-growing tumors) or 20 days(slowly-growing tumors). Tumors and normal tissues were excisedimmediately and snap-frozen over liquid nitrogen. Normal tissues werealso harvested from non-tumor-bearing animals. Antibody localizationstudies were performed in animals bearing 1 cm subcutaneous tumorsinduced by injection of C1300 and C1300(Muγ) in the ratio 7:3. Onehundred micrograms of unconjugated M5/114 or CAMPATH-2 antibodies or 100μg biotinylated 11-4.1 or WT-1 antibodies in 100 μl PBS were injectedintravenously. At various times thereafter the animals were euthanizedand their circulation was flushed with PBS for 5 minutes before removaland freezing of tumors and normal tissues as before. 8 μM frozensections were cut on a Tissuetek 2 cryostat (Baxter) and air-dried for 2hours at room temperature. Slides were stored at -20° C. for up to 3months before assay.

Indirect immunoperoxidase staining for rat IgG was adapted from a methoddescribed by Billington et al. (1986). Sections were allowed to returnto room temperature, air dried for 30 minutes and fixed in acetone for15 minutes. After rehydration in PBS for 5 minutes, sections wereincubated in a humidified chamber for 45-60 minutes with primaryantibodies, diluted optimally in PBS-0.2% BSA, (PBS-BSA). After twowashes in PBS, the sections were incubated for 30-45 minutes withhorseradish peroxidase-conjugated rabbit anti-mouse IgG (Dakopatts,Carpinteria, Calif.) diluted 1:10 in PBS-BSA supplemented with 20%normal mouse serum (ICN, High Wycombe, UK) to block antibodiescross-reacting with mouse immunoglobulins. After a further two washes inPBS, the reaction product was developed using 0.5 mg/ml3',3'-diaminobenzidine (Sigma) containing 0.01% (v/v) hydrogen peroxidefor 8 minutes. The sections were counterstained with Mayer's hematoxylin(Sigma) for 15 seconds, dehydrated in absolute ethanol, cleared inxylene and mounted with Accumount 60 medium (Baxter). Indirectimmunoperoxidase staining with biotinylated mouse antibodies was carriedout in the same manner, except that peroxidase-conjugated streptavidin-biotin complex, diluted 1:50 in PBS with no blocking serum, was used asthe second layer.

B. Results

1. Murine IFN-γ levels in C1300(Muγ) conditioned-medium

C1300(Muγ)-conditioned medium contained 50.2-63.5 I.U./ml murine IFN-γ,in accordance with previous reports (Watanabe et al., 1989). Bycontrast, less than 5 I.U./ml IFN-γ was detected in C1300-conditionedmedium or C1300(Muγ)-conditioned medium to which an excess ofneutralizing anti-IFN-γ antibody had been added 24 hours before assay.

2. Induction of MHC Class II (I-E^(k)) on SVEC cells by r.IFN-γ inC1300(Muγ)-conditioned-medium

As shown in FIG. 1a, unstimulated SVEC cells did not express I-E^(k). Bycontrast, a large majority of cells preincubated with r.IFN-γ (FIG. 1a)or with C1300(Muγ)-conditioned medium (FIG, 1b) expressed significantlevels of I-E^(k), and this induction was almost completely blocked byanti-IFN-γ. Treatment of SVEC cells with r.IFN-T or C1300(Muγ)-conditioned medium did not cause non-specific antibody bindingsince the isotype-matched control antibody did not bind to the cells.These results were confirmed by indirect immunoperoxidase staining ofcytospin preparations.

These findings suggested that vascular endothelial cells in tumorscontaining sufficient quantities of IFN-γ-secreting C1300(Muγ) cellsshould be induced to express high cell surface levels of MHC Class IImolecules.

3. Expression of MHC Class I (H-2K^(k)) and Class II (I-E^(k)) by C1300and C1300(Muγ) cells

Since IFN-γ can induce MHC Class II antigen expression in diverse celltypes (Capobianchi et al. 1985; Collins et al., 1984; Hokland et al.,1988) and since the M5/114 antibody crossreacts with I-E^(k), wedetermined whether the M5/114 antibody intended for use to target tumorendothelial cells in vivo would also bind to the tumor cells themselves.As shown in FIG. 2a, C1300(Muγ) cells expressed I-E^(k), but at levels10-20 fold lower than those on SVEC cells stimulated with IFN-γ.

Similarly, C1300 cells expressed detectable but low levels of H-2K^(k)whereas C1300(Muγ) cells displayed uniformly high levels, approximately20-fold greater than on the parental line (FIG. 2b). This result wasexpected from the known autocrine Class I-inducing activity of IFN-γ andis in keeping with a previous report (Watanabe et al., 1989). Cocultureof C1300(Muγ) cells and C1300 cells induced homogeneous expression ofI-E^(k) and H-2K^(k) on both populations (FIG. 2). Induction of theseantigens on C1300 cells appears to be caused by IFN-γ released into theculture medium by the C1300(Muγ) cells since the effect was centralizedby anti-IFN-γ antibodies.

4. Growth of C1300 and C1300 (Muγ) tumors in immunodeficient mice andinduction of Ia^(d) on tumor vascular endothelial cells

The inventors first attempted to grow subcutaneous C1300(Muγ) tumors inBALB/c nu/nu and SCID mice because both strains carry the MHC haplotype(H-2^(d)) with which the anti-MHC Class II antibody M5/114 reacts, andbecause neither strain would be expected to reject the tumors, as dosyngeneic immunocompetent A/J animals (Watanabe et al., 1989). Forunknown reasons inocula composed entirely of C1300(Muγ) cells failed toproduce progressively-growing tumors in BALB/c nu/nu or SCID mice.Conversely, pure C1300 inocula displayed 100% tumorigenicity but, asexpected, did not contain Ia^(d) -positive endothelial cells.

In order to identify a combination which would yield a high percentageof tumor takes, reliable growth kinetics and cause Ia^(d) induction of alarge majority of intratumoral endothelial cells, several ratios ofC1300 and C1300(Muγ) cells were inoculated into BALB/c nu/nu mice. Asshown in FIG. 3, mixtures containing C1300 and C1300(Muγ) cells in theratio 9:1 produced rapidly-growing tumors but, when sections of thetumors were stained with anti-Ia^(d) antibody by the indirectimmunoperoxidase technique, none of the endothelial cells in the tumorwere found to be stained. Dropping the ratio of C1300: C1300(Muγ) to 8:2gave rapidly-growing tumors in which approximately 50% of blood vesselswere Ia^(d) -positive. Dropping the ratio further to 7:3 or 5:5 producedtumors which grew quite rapidly and contained a large majority of Ia^(d)-positive vessels. Dropping the ratio still further to 3:7 producedtumors in no more than half of the animals and those tumors that becamepalpable failed to grow beyond 6 mm in diameter. Histological analysesof the latter revealed no morphologically recognizable intact bloodvessels and, hence, it was not possible to ascertain their level ofIa^(d) expression.

Of the two usable C1300: C1300(Muγ) ratios identified, 7:3 and 5:5, theratio of 7:3 was adopted for the remainder of this study because thetake rate was higher (100% vs. 80%) and the variability in tumor growthrate between individual animals was lower.

5. Distribution of Ia^(d) in BALB/c nude and SCID mice

The distribution of M5/114 binding in tissues from tumor-bearing BALB/cnu/nu mice is shown in Table III. In subcutaneous tumors, most or allvascular endothelial cells and numerous interstitial macrophages werestained. In most organs, the binding of M5/114 reflected the classicaldistribution of MHC Class II antigens, being restricted to B cells inlymphoid organs, resident macrophages in all tissues studied exceptbrain and to tissue-specific elements of the reticuloendothelial system,such as liver Kupffer cells and Langerhans cells of the skin. Inaddition, staining was occasionally seen in some kidney tubules. Whensections of small and large intestine from BALB/c nu/nu mice wereexamined, heavy labeling of both epithelial and endothelial cells wasseen in both regions. By contrast, very little staining with M5/114 wasseen in sections of intestine from SCID mice maintained in germ-freeconditions. The staining of nu/nu mouse intestine was found to berelated to the microbiological status of the animals and is discussedbelow. Apart from in the gut, no staining of endothelial cells withM5/114 was seen in any tissues examined in either nu/nu or SCID mice.The distribution of Ia^(d) antigens in normal tissues was not affectedby the presence of the tumor because the staining pattern of M5/114 wasidentical in non-tumor-bearing mice.

                                      TABLE III                                   __________________________________________________________________________    Localization of Intravenously Administered anti-1a.sup.(d) Antibody            in C1300(MUγ) Tumor-Bearing Mice..sup.(a)                                                      Localization in vivo                                  Tissue Antigen Expression                                                                             1 hour                                                                             4 hours                                                                            24 hours                                    __________________________________________________________________________    Tumor.sup.(b)                                                                        Endothelial cells (EC), Mφ                                                                 EC.sup.(c)                                                                         EC   EC.sup.(d)                                    Brain None None None None                                                     Colon.sup.(a) Minority of epithelium & EC, Mφ None None None                                               Duodenum Some epithelial cells & EC,                                         Mφ None None None                         Heart Interstitial Mφ None None None                                      Kidney Occasional proximal tubule, Mφ None None None                      Liver Kupffer cells (KC), numerous Mφ in KC.sup.(c) KC KC.sup.(d)                                           parenchyma   some Mφ                    Lung Numerous Mφ in parenchyma None None None                             Pancreas Numerous Mφ in parenchyma None None None                         Skin.sup.(e) Langerhans cells None None None                                  Spleen Red pulp (RP) Mφ, marginal zone (MZ) MZ MZ MZ, RP                   B cells & Mφ, some T/B cells in PALS  PALS PALS                        __________________________________________________________________________     .sup.(a) Studies performed with SCID or antibiotictreated BALB/c nu/nu        mice.                                                                         .sup.(b) Mixed tumor of 7:3 C1300:C1300(Muγ) cells grown                subcutaneously.                                                               .sup.(c) Strong staining, including discemable labelling of luminal           membranes.                                                                    .sup.(d) Weaker staining, entirely intracellular.                             .sup.(e) Either adjacent to, or distant from tumor.                           PALS: Periarteriolar lymphatic sheath, Mφ: Macrophages               

6. Attenuation of expression of Ia^(d) on colonic endothelium andepithelium of nude mice by administration of antibiotics

In BALB/c nu/nu mice, most epithelial cells from all regions of the gutwere intensely stained with anti-Ia^(d) antibody. In addition, someendothelial cells in both upper and lower bowel bound M5/114 antibody,particularly those associated with colonic villi. When the animals weretreated with oral tetracycline-Hcl, a broad-spectrum antibiotic, for 1-3weeks there was a progressive diminution of Ia^(d) expression in thecolon and elsewhere in the gut, so that binding of M5/114 was in mostsections restricted to the luminal membranes of a minority of epithelialcells. Light cytoplasmic staining of occasional endothelial cells wasobserved in some antibiotic-treated animals. The pattern of epithelialand endothelial Ia^(d) expression was not homogeneous and the intensityof M5/114 staining correlated with the frequency of CD3+ T lymphocytesin the adjacent lamina propria. Antibiotic treatment was associated witha dramatic decrease in the numbers of intravillous CD3-positive cells:after three weeks practically all had disappeared from the underlyingparenchyma and associated lymphoid deposits and there was a coincidentdecline in Iad expression on surrounding epithelial and endothelialcells.

In SCID mice, epithelial and endothelial cell Ia^(d) expression andT-cell infiltration of the colon resembled that of antibiotic-treatedBALB/c nu/nu animals.

7. Specific localization of intravenously administered anti-Ia^(d)antibody to tumor vasculature, B cells and macrophages in SCID andantibiotic-treated nude mice

Tumor-bearing BALB/c nu/nu and SCID mice were given intravenousinjections of anti-Ia^(d) or the isotype-matched control antibody andeuthanized 1, 4 or 24 hours later. The in vivo localization ofanti-Ia^(d) antibody in tumor and normal tissues is shown in Table III.Anti-Ia^(d) antibody was found on the luminal membrane and in thecytoplasm of most or all tumor vascular endothelial cells one hour afterinjection. A similar pattern was seen at four hours after injection, butby 24 hours the labeling of tumor endothelial cells was weaker andentirely intracellular, consistent with the progressive internalizationand metabolism of the antibody by endothelial cells (Table III). Also,at 24 hours small amounts of antibody were detectable in the immediateperivascular regions of the tumor.

Anti-Ia^(d) antibody was bound to Kupffer cells in the intravascularcompartment of the liver within one hour of injection. At later timesafter injection, internalization and degradation of the antibody wasapparent (Table III). Adjacent sinusoidal endothelial cells were notstained. The high permeability of hepatic fenestrated endothelia wasindicated by the penetrance of the antibody to reach some hepaticparenchymal macrophages (Table III). In the spleen, perivascular B cellsand macrophages in white pulp marginal zones were stained within onehour, showing that the vasculature of this organ was particularlypermeable to antibody. At later stages the antibody penetratedthroughout the splenic lymphoid compartment and also labelled a minorityof red pulp macrophages (Table III). In organs other than the liver andspleen, macrophages and related cells such as the Langerhans cells ofthe skin were unstained probably because their vascular endotheliumcontains tight junctions and is relatively impermeable to antibodies.

Anti-Ia^(d) antibody was bound to some endothelial cells in the colon ofBALB/c nu/nu mice, but not elsewhere in the intestine, one hour afterinjection. Antibiotic treatment for 1-3 weeks before injection ofanti-Ia^(d) antibody completely abolished localization to gutendothelial cells. No intravenously injected anti-Ia^(d) antibody homedto gut endothelia in SCID mice. The isotype-matched control antibody wasnot detected in tumor or normal tissues at any time after injection.

Taken together, these results strongly indicate that, when injected intoappropriate tumor-bearing animals anti-Ia^(d) antibody orimmunoconjugates will localize effectively to most or all tumorendothelial cells while sparing life-sustaining normal tissues.

8. Perivascular staining of tumor cells in mice injected with anti-tumor(H-2K^(k)) antibody

When frozen sections of subcutaneous tumors deriving from inocula ofmixed C1300 and C1300(Muγ) cells (7:3) were stained with biotinylatedanti-H-2K^(k) antibody, a homogeneous staining pattern was obtained. Thelevels of IFN-γ secreted by the C1300(Muγ) cells in the tumor weretherefore sufficient to induce increased H-2K^(k) expression by theC1300 component of the tumor, in accordance with the in vitro co-culturestudies described above. The staining was specific because no stainingwas seen with the isotype-matched control antibody. No specific labelingof any normal tissue by anti-H-2K^(k) antibody was found, as expectedsince this antibody was raised in an H-2d mouse strain.

In contrast with the rapid binding of intravenously-administeredanti-Ia^(d) antibody to tumor vasculature, no significant accumulationof anti-H-2K^(k) antibody was apparent one hour after injection. Afterfour hours, however, anti-H-2K^(k) antibody was detected in smallislands of tumor cells surrounding central capillaries. After 24 hours,the antibody was bound to larger discrete areas of tumor cells butstaining intensity was diminished relative to the earlier time points.Each with localization times of up to 72 hours, homogenous labeling ofall tumor cells was not achieved.

No localization of anti-H-2K^(k) antibody was found in any normaltissues and binding of the isotype-matched control antibody was notdetectable in tumor or normal tissues.

C. Discussion

This example describes a murine model for studying the antibody-directedtargeting of vascular endothelial cells in solid tumors. In this model,IFN-γ gene-transfected tumor cells growing in SCID or antibiotic-treatednude mice release IFN-γ which induces the de novo expression of MHCClass II antigens on the tumor vasculature. MHC Class II is absent fromthe vasculature in the normal tissues of these mice and hence the ClassII induced on the tumor vascular endothelial cells serves as a specificmarker. Class II is present on B-lymphocytes, Kupffer cells and othercells of monocyte/macrophage lineage but these cells are notlife-sustaining so their temporary absence after targeting withcytotoxic immunoconjugates should be tolerable. IFN-γ also induces thetumor cells themselves to express high levels of the MHC Class Iantigen, H-2K^(k), which can serve as a tumor cell-specific marker inBALB/c nu/nu or SCID mice, which both carry the H-2K_(d) haplotype.Thus, anti-Ia^(d) and anti-H-2K^(k) antibodies injected systemicallylocalize selectively to tumor vascular endothelial cells and tumor cellsrespectively, which enables the approaches of targeting the tumorvasculature and the tumor cells to be compared in this model, or used incombination.

It was necessary to dilute the C1300(Muγ) cells with C1300 parentalcells in the ratio 3:7 to establish progressively-growing subcutaneoustumors in which the vascular endothelial cells were Class II(lad)--positive. Undiluted C1300(Muγ) cells were poorly tumorigenic inBALB/c nu/nu mice, in contrast with a prior report (Watanabe et al.,1989). Vascular dysfunction appeared to be the reason why pureC1300(Muγ) tumors would not grow beyond a diameter of 5-6 mm. Stainingof sections of tumors with the anti-endothelial cell antibody MECA 20revealed that the vessels were morphologically atypical with no visiblelumens. It is possible that excessively high intratumoral IFN-γ levelsin pure C1300(Muγ) tumors caused direct vascular toxicity or activatedmacrophages in the tumor to become cytotoxic for endothelial cells (Periet al., 1990).

Intravenously injected anti-Ia^(d) antibody bound rapidly andhomogeneously to vascular endothelial cells in the tumor, confirming theimmediate accessibility of intravascular targets (Kennel et al., 1991).Remarkably, the inductive influence of IFN-γ from C1300(Muγ) cells wascompletely restricted to the tumor mass: endothelial cells in theoverlying area of skin expressed no detectable Ia^(d) and did not bindany intravenously-injected anti-Ia^(d) antibody. It is likely that IFN-γentering the systemic circulation is neutralized by a specific bindingprotein, perhaps a soluble form of the IFN-γ receptor (Novick et al.,1989), whose normal role may be to down-regulate cytokine activity(Fernandez-Botran et al., 1991) or to restrict it to the immediatelocale of secretion.

Ia^(d) antigens are not restricted solely to tumor endothelial cells.MHC Class II antigens are expressed constitutively by B cells, activatedT cells and cells of the monocyte/macrophage lineage in humans androdents (Daar et al., 1984; Hammerling et al, 1976) and were found inthis study also to be present on occasional proximal tubules in thekidney and on some epithelial cells in the intestine of SCID andantibiotic-treated BALB/c nu/nu mice. However, when injectedintravenously, only the hepatic Kupffer cells, splenic B cells andmacrophages in the liver and spleen bound detectable amounts of theanti-Ia^(d) antibody: the potentially life-sustaining Class II--positiverenal and gut epithelial cells were unstained. Localization ofintravenously-injected anti-Ia^(d) antibody to hepatic Kupffer cells andsplenic marginal zone B cells occurred within one hour, in accordancewith the report of Kennel et al. (1991). Presumably, the extremepermeability of the discontinuous splenic endothelium permits rapidextravasation of antibodies into the parenchyma of this organ andstaining of the marginal zone B-cells (Kennel et al., 1991).

The reason for the lack of staining of renal and gut epithelial cells isprobably that these cells are not readily accessible tointravenously-administered antibody because the antibody would have todiffuse across basement membranes and several tissue layers to reachthese cells. In addition, it is likely that all the remaininganti-Ia^(d) antibody in the circulation was absorbed by more accessiblesplenic white pulp lymphocytes before significant extravasation into thered pulp (Kennel et al., 1991; Fujimori et al., 1989) or other normaltissues could occur. This is important because it illustrates apotentially critical pharmacokinetic difference between vasculartargeting and tumor cell targeting. Because the tumor endothelial cellsare so accessible to intravenously-administered antibody, the presenceof a large `sink` of competing antigen in the blood or lymphoid organsshould not prevent the antibody from reaching the target cells butshould protect antigen-positive cells in most extravascularcompartments. It is conceivable that an antibody recognizing a tumorvascular endothelial cell antigen that is shared by epithelial cells,for instance, might be targeted without the toxic side-effects whichhave complicated therapy with anti-tumor cell immunoconjugates (Spitler,1988). Furthermore, even in the absence of such a sink, it is possiblethat operative specificity for tumor endothelial cells could be achievedin the face of cross-reactivity with extravascular normal tissues bydecreasing the dose or by using rapidly-cleared antibody fragments inthe construction of the immunoconjugate.

Although anti-Ia^(d) antibody did not localize to life-sustainingIa^(d) + extravascular tissues such as kidney tubules and gutepithelium, it did bind to colonic endothelial cells innon-antibiotic-treated BALB/c nu/nu mice. These cells were as accessibleas tumor endothelial cells and were required for survival since regularBALB/c nu/nu mice treated with high doses of M5/114 immunotoxins diedfrom intestinal damage. Murine endothelial cells do not express MHCClass II antigens in vitro (O'Connell et al., 1990; Duijvestijn et al.,1986) or in vivo (de Waal et al., 1983) unless stimulated with IFN-γ soit is likely that induction of Ia^(d) on intestinal endothelial andepithelial cells was a result of local secretion of IFN-γ by helper Tcells (Cherwinski, 1987) or activated NK cells (Anegon, 1988; Kasahara,1983) in response to gut flora. In accordance with this view, numerousCD3+, CD8+ T cells were observed in the villous stroma and theirfrequency correlated with the intensity of staining of endothelial andepithelial cells with anti-Ia^(d) antibody. Furthermore, oraladministration of tetracycline-Hcl (a broad spectrum antibiotic)reversed T cell infiltration, diminished Ia^(d) expression and abolishedlocalization of intravenously-injected anti-Ia^(d) antibody to colonicendothelial cells.

Antibiotic treatment had no effect on Ia^(d) expression by tumorendothelial cells. In subsequent studies it was found that SCID mice hadlittle Ia^(d) on colonic epithelial or endothelial cells and thatintravenously-administered anti-Ia^(d) antibody did not localize totheir colonic endothelium. Furthermore, high doses of M5/114immunotoxins were non-toxic in these animals. Given the possibility ofantibiotic resistance arising in the gut flora of tetracycline-treatedBALB/c nu/nu mice, we believe that SCID mice may be more suitable forthese types of studies.

Consistent with the findings of others (Baxter et al., 1991; Kennel etal., 1991; Jones et al., 1988; Pervez et al., 1988), an anti-tumorantibody directed against the H-2K^(k) antigen on C1300 and C1300(Muγ)cells showed perivascular staining of tumor cells after intravenousadministration. In view of the homogeneous expression of H-2K^(k) bytumor cells in vitro and in sections of subcutaneous tumors, it islikely that the uneven intratumoral distribution ofintravenously-injected anti-H-2K^(k) antibody was related to thevascular and interstitial physiology of the tumor (Jain, 1990; Fujimoriet al., 1989). This nicely demonstrates, in a single system, thelimitations of using antitumor antibodies for targeting and the virtuesof tumor vascular targeting. It may be possible to combine bothapproaches to advantage because the tumor cells that survive destructionof intratumoral blood vessels are likely to be those at the periphery ofthe tumor mass, close to the tumor-host interface. These areas arelikely to be well vascularized by capillaries in adjacent normal tissuesand have low interstitial pressure (Jain, 1990), so the surviving cellsshould be amenable to attack by antitumor immunoconjugates.

In summary, the inventors describe a murine model with which to test thefeasibility of targeting the vasculature of solid tumors. The modelpermits the antitumor effects of immunoconjugates directed against tumorvasculature to be compared with those of immunoconjugates directedagainst the tumor cells themselves.

EXAMPLE II Solid Tumor Therapy Using A Vascular Targeted Iunmunotoxin

This example describes the successful therapy of the solid tumor modeldescribed in Example I, using the anti-tumor endothelial cellimmunotoxin, MS/114dgA, and the anti-tumor cell immunotoxin, 11-4.1dgA,alone as well as in combination therapy.

A. Materials and Methods

1. Animals

BALB/c nu/nu mice were purchased from Simonsen (Gilroy, Calif.). SCIDmice were from the National Cancer Institute (Bethesda, Md.). Germ-freeSCID mice were from the University of Wisconsin (Madison, Wis.). Allanimals were maintained in microisolation units on sterilized food andwater.

2. Cells and Culture Conditions

All cell lines used in this study were cultured in modified Eagle'smedium (MEM) supplemented with 10% (v/v) fetal calf serum, 2.4 mML-glutamine, 200 units/ml penicillin and 100 μg/ml streptomycin.Cultures were maintained at 37° C. in a humidified atmosphere of 90%air/10% CO₂. The C1300 neuroblastoma line was established from aspontaneous tumor which arose in an A/Jax mouse in 1940 (Dunham et al.,1953). The C1300(Muγ)12 line, hereafter abbreviated to C1300 (Muγ), wasderived by transfection of C1300 cells with murine IFN-γ gene using theIFN-γ expression retrovirus PSVX (MuγδA_(s)) (Watanabe et al., 1988),and was cultured in MEM as above containing 1 mg/ml G418 (Geneticin,Sigma). Both lines carry the MHC haplotype H-2K^(k), I-A^(k), I-E^(k),D^(d). C1300 and C1300(Muγ) cells were grown in regular tissue cultureflasks or, when large quantities were required for in vivo studies incell factories (Baxter, Grand Prairie, Tex.). Cells from subcutaneoustumors were recovered for in vitro analysis by gentle mincing in MEM.After tumor cells had adhered overnight the monolayers were washed twicewith MEM to remove nonadherent contaminant host cells.

Tumor conditioned media were prepared by seeding C1300 and C1300(MuAγ)cells at 25% of confluent density and culturing them for four days.Conditioned media were dialyzed for 16 hours against MEM without FCS toremove G418, filtered through a 0.22 μM membrane and stored at 4° C. forno more than one week before assay. Aliquots of anti-IFN-γ antibodies(see `Monoclonal Antibodies`) sufficient to neutralize 200 internationalunits (I.U.) of murine IFN-γ/ml of conditioned medium were added to somesamples 24 hours before assay. The SVEC-10 murine endothelial cell line,hereafter abbreviated to SVEC, was kindly provided to Dr. M. Edidin,Department of Biology, Johns Hopkins University, Baltimore, Md. and wasderived by immortalization of lymph node endothelial cells from a C3H(H-2^(k)) mouse with SV40 (O'Connell et al., 1990). For some studies,SVEC cells were cultured for 72 hours with 100 I.U./ml recombinantmurine IFN-γ, (r.IFN-γ, obtained from Dr. F. Balkwill, Imperial CancerResearch Fund, London, England) or tumor-conditioned medium. Inaddition, 200 I.U./ml anti-IFN-γ antibody was added to some flasks atthe beginning of the 72 hour culture period.

3. Monoclonal Antibodies

The M5/114.15.2 (hereafter abbreviated to M5/114) and 11-4.1 hybridomaswere purchased from the American Type Collection (Rockville, Md.) andwere grown in MEM-10% FCS. The antibodies were purified from culturesupernatant by precipitation in 50% ammonium sulphate and affinitychromatography on Protein A. The rat IgG2b antibody, M5/114, detects anIa specificity on I-A^(b), I-A^(q), I-A^(d), I-E^(d) and I-E^(k)molecules (Bhattacharya et al., 1981). Thus, the antibody recognizesI-E^(k) molecules on SVEC (H-2^(k)) cells and I-A^(d) and I-E^(d),hereafter referred to collectively as Ia^(d), on cells from BALB/C nu/nuor SCID mice (both H-2^(d)). The mouse IgG2a antibody 11-4.1 recognizesH-2K^(k) but not H-2K_(d) molecules (Oi et al., 1978) and so binds toH-2K^(k) on C1300 and C1300(Muγ) cells but is unreactive with MHCantigens from BALB/c nu/nu or SCID mice. Isotype-matched controlantibodies of irrelevant specificity were CAMPATH-2 (rat IgG2b,anti-human CD7 (Bindon, 1988) and WT-1 (mouse IgG2a, anti-human CD7 (Taxet al., 1984). Purified preparations of CAMPATH-2 and WT-1 were obtainedfrom Dr. G. Hale (Department of Pathology, Cambridge, England) and Dr.W. Tax (Sint Radboudzeikenhuis, Nijmegen, the Netherlands) respectively.

4. Preparation of dgA

The ricin A chain was purified by the method of Fulton et al. (Fulton etal., 1986). Deglycosylated ricin A was prepared as described by Thorpeet al. (1985). For conjugation with antibodies, the A chain was reducedwith 5 Mm DTT and subseqvuently separated from DTT by gel filtration ona column of Sephadex G-25 in PBS, pH 7.5.

5. Preparation of Immunotoxins

IgG immunotoxins were prepared using the4-succinimidyloxycarbonyl-amethyl(1-pyridyldithio)toluene linking agentdescribed by Thorpe et al. (1987).4-succinimidyloxycarbonyl-α-methyl(2-pyridyldithio)toluene dissolved indimethylformamide was added to the antibody solution (7.5 mg/ml inborate buffer, pH 9.0) to give a final concentration of 0.11 Mm. After 1hour the derivatized protein was separated from unreacted material bygel chromatography on a Sephadex G-25 column and mixed with freshlyreduced ricin A chain. The solution was concentration to about 3 mg/mland allowed to react for 3 days. Residual thiol groups were inactivatedby treating the immunotoxin with 0.2 Mm cysteine for 6 hours. Thesolution was then filtered through a Sephacryl S-200 HR column in 0.1Mphosphate buffer, pH 7.5, to remove unreacted ricin A, cysteine, andaggregates. Finally, the immunotoxin was separated from free antibody bychromatography on a Blue Sepharose CL-6B column equilibrated in 0.1Msodium phosphate buffer, pH 7.5, according to the method of Knowles andThorpe (1987).

6. Cytotoxicity Assays

C1300, C1300(Muτ) and SVEC cells suspended at 10⁵ cells/ml in MEM-10%FCS were distributed in 100 μl volumes into the wells of flat-bottomedmicrotiter plates. For some assays, SVEC cells were suspended in C1300-or C1300(Muτ) -conditioned medium or MEM supplemented with 100 I.U./mlr.IFN-τ as indicated. Immunotoxins in the same medium were added (100μl/well) and the plates were incubated for 24 hours at 37° C. in anatmosphere of 10% CO₂ in humidified air. After 24 hours, the cells werepulsed with 2.5 μCi/well [³ H] leucine for another 24 hours. The cellswere then harvested onto glass fiber filters using a Titertek harvesterand the radioactivity on the filters was measured using a liquidscintillation spectrometer (LKB; Rackbeta). The percentage of reductionin [³ H] leucine incorporation, as compared with untreated controlcultures, was used as the assessment of killing.

7. Antitumor Studies

For the establishment of solid tumors, a mixture of 1.4×10⁷ C1300 cellsand 6×10⁶ C1300(Muγ) cells in 200 μl MEM-30% FCS were injectedsubcutaneously into the right anterior flank of BALB/c nu/nu or SCIDmice. Fourteen days later, when the tumors had grown to 0.8-1.2 cm indiameter, the mice were separated into groups of 5-10 animals andinjected intravenously with 200 μl of immunotoxins, antibodies ordiluent. Perpendicular tumor diameters were measured at regularintervals and tumor volumes were estimated according to the followingequation (Steel, 1977).

    volume--Smaller diameter.sup.2 ×larger diameter×π/6

For histopathological analyses, animals were euthanized at various timesafter treatment and the tumors were excised immediately into 4% (v/v)formalin. Paraffin sections were cut and stained with hematoxylin andeosin or Massons trichrome.

B. Results

The first studies carried out involved a comparison of killing activityof anti-Class II immunotoxin (M5/114 dgA) against unstimulated SVECmouse endothelial cells with those stimulated with conditioned mediumfrom IFN-γ-secreting tumor cells (Cl300 Muγ). These studies were carriedout in order to demonstrate that the anti-Class II immunotoxin, M5/114dgA exerts a selective toxicity against IFN-γ stimulated endothelialcells, and not against unstimulated cells. The results are shown inFIGS. 4a and b. In FIG. 4a, SVEC mouse endothelial cells were culturedin regular medium and the cultured cells subjected to varyingimmunotoxin concentrations as indicated. As will be appreciated, whilericin effected a 50% inhibition of leucine incorporation at about3×10⁻¹¹, neither the anti-Class II immunotoxin (M5/114 dgA) nor thecontrol immunotoxin (CAMPATH-2 dgA) exerted a significant toxic effectwithin the concentration ranges tested. In contrast, when the SVEC mouseendothelial cells were stimulated by culturing in the presence ofC1300(Muγ)-conditioned medium, the mouse endothelial cells became quitesensitive to the anti-Class II immunotoxin, with 50% of stimulated cellsbeing killed by the anti-Class II immunotoxin at a concentration ofabout 3×10-¹⁰ M. Thus, these studies demonstrate that γ interferon,which is produced by the C1300(Muγ) and present in the conditioned mediaeffectively promote the appearance of Class II targets on the surface ofthe SVEC cells.

FIG. 5 illustrates similar studies, which confirm the finding that theC1300(Muγ) conditioned media effectively promotes the expression ofClass II molecules on endothelial cells. In particular, the data shownin FIG. 5 demonstrate that both recombinant IFN-γ as well as conditionedmedia from C1300(Muγ) sensitize endothelial cells to the anti-tumorendothelial cell immunotoxin, M5/114 dgA. FIG. 5 also demonstrates thatconditioned media from C1300 cells that do not secrete interferon (C1300TCM), as well as interferon-producing C1300 cells (Muγ) pretreated withanti-IFN-γ, both do not promote an anti-Class II immunotoxin sensitizingeffect.

Next, a series of studies were carried out wherein the killing activityof an anti-Class I (anti-tumor) immunotoxin (11-4.1-dgA) and that of theanti-Class II immunotoxin (M5/114-dgA) are compared against a 70:30mixed population of C1300 and C1300(Muγ) cells. FIG. 6a simplydemonstrates that in a 70:30 culture of C1300 and C1300(Muγ), that onlythe anti-Class I immunotoxin, 11-4.1-dgA, and ricin, exert a cytotoxiceffect. FIG. 6b shows killing of cells freshly recovered fromsubcutaneous tumors in mice. Taken together, these FIGS. demonstratethat the anti-tumor immunotoxin kills tumor cells well, but that theanti-tumor endothelial cell immunotoxin does not. Thus, any anti-tumoreffect of M5/114-dgA would not likely be due to direct tumor cellkilling. Therefore, these studies serve as a control for later studieswherein it is demonstrated that M5/114-dgA can have a profoundanti-tumor effect in the solid tumor model system described in ExampleI, through an anti vascular effect.

FIG. 7 also shows a comparison of killing of pure and mixed populationsof C1300 and C1300(Muγ) by the anti-tumor cell immunotoxin 11-4.1 dgA(anti-H-2K^(k)). Both FIGS. show the effects of the anti-tumor cellimmunotoxin against four different tumor populations. Again, in eachcase the anti-tumor cell immunotoxin demonstrate significant anti-tumoractivity, at a concentration of on the order of about 10⁻¹⁰ M. Thus,these data show that mixed tumors should be highly sensitive to theanti-tumor immunotoxin, a control that is needed in order to demonstratethe anti-vascular attributes of the anti-Class II immunotoxin.

The next series of studies involved the application of one or both ofthe foregoing anti-Class I and anti-Class II immuno-toxins, in the modeltumor system disclosed in Example I. FIG. 8 illustrates the anti-tumoreffects of the anti-tumor endothelial cell immunotoxin, M5-114 dgA. Ascan be seen, dosages as low as 20 μg exhibited a noticeable antitumoreffect. While the change in mean tumor volume in the 20 μg-treatedpopulation does not, in FIG. 8, appear to be particularly dramatic,sections of the tumor, when H & E-stained, illustrated surviving"islands" of tumor cells in a "sea" of necrotic cells. This can be seenin FIG. 9, wherein the surviving islands of tumor cells are the darkerstaining areas, and the necrotic tissue the more lightly-staining areas.

Importantly, treatment with 40 μg of M5/115-dgA resulted in dramaticanti-tumor effects, as can be seen in FIG. 8. Here, 30 days after tumorinoculation the mean tumor volume equated with the 16 day figure in thecontrols. The dotted line in FIG. 8 represents the results that wereexpected with the use of 100 μg of M5/114 dgA, with a possiblereoccurrence of tumor cell indicated at the 26 day position being thepartial result of a surviving rim of viable cells observed in thetreated solid tumor.

FIG. 10 is a section through a 1.2 cm tumor 72 hours after treatmentwith 100 μg of the anti-Class II immunotoxin M5/114 dgA, followed by H &E staining. As can be seen, this pattern is similar to the 20 μg datashown in FIG. 9, but certainly much more dramatic in that virtually no"islands" of tumor cells remain. It is estimated that this patternrepresents a complete necrosis of greater than 95% of the tumordiameter, leaving only a thin cuff of surviving tumor cells, presumablynourished by vessels in overlying skin.

To address this potential source of recurrence, i.e., the potential fora cuff of surviving tumor cells, combined therapy with both an antitumor(anti-Class I) and an anti-endothelial (anti-Class II) immunotoxin wasundertaken. The theory for this combined therapeutic approach can beseen in FIG. 11, which illustrates the appearance of the solid tumorfollowing 48-72 hours of intravenous immunotoxin treatment. At the lefthand side of the FIG. is represented a tumor following anti-tumorimmunotoxin therapy alone. As illustrated, only those areas immediatelysurrounding the blood vessels become necrotic following treatment withthe anti-tumor immunotoxin, due to the inability of the immunotoxin tosufficiently infiltrate the tumor and reach the tumor cells that aredistal of the blood vessels. In stark contrast, shown in the middlepanel of FIG. 11 is a representation of the low dose treatment withanti-endothelial cell immunotoxin. Here is illustrated the effects of alow dose of the anti-endothelial cell immunotoxin, which results innecrosis of the tumor in those parts distal of the blood vessels, exceptfor the outer rim of the tumor which is presumably fed by associatednormal tissues. At low dosages, only those areas of the tumor closest tothe blood vessels will receive sufficient oxygen and nutrients. Next,the high dose anti-endothelial immunotoxin results are illustrated onthe right hand side of FIG. 11. Here, the only living tumor remaining isthat associated with the outer rim of the tumor. It was a goal ofcombined therapy studies to demonstrate an additive or even synergisticeffect when both an anti-tumor immunotoxin and anti-endothelial cellimmunotoxin were employed in combination. This effect is illustrated inthe panel at the right hand side of FIG. 11.

The results of this combination therapy are shown in FIG. 12. FIG. 12shows the anti-tumor effects of the anti-tumor immunotoxin (11-4.1-dgA)alone at a high dose, the anti-tumor endothelial cell immunotoxin(M5/114-dgA) alone at a low dose, as well as combinations of both. Theresults demonstrate that both immunotoxins had a transient butnoticeable effect in and of themselves, with the anti-tumor immunotoxinshowing a slightly greater anti-tumor effect than the anti-tumorendothelial cell immunotoxin, although this might be a dosing effect.

Truly dramatic synergistic results were seen when both were used incombination. When 100 μg of the anti-tumor immunotoxin was given on day14, followed by 20 μg of the anti-tumor endothelial cell immunotoxin onday 16, one out of four cures were observed. When the order ofadministration was reversed, i.e., the anti-tumor endothelial cellimmunotoxin given first, even more dramatic results were observed, withtwo out of four cures realized. The latter approach is the more logicalin that the initial anti-endothelial cell therapy serves to remove tumormass by partial necrosis, allowing better penetration into the tumor ofthe anti-tumor immunotoxin.

Therapeutic doses (≧40 μg) of M5/114-dgA did not cause detectable damageto Class II-positive epithelial cells or to hepatic Kupffer cells, asassessed by histopathological analysis at various times after treatment.Any lymphoid cells destroyed by M5/114-dgA were apparently replaced frombone marrow precursors because, 20 days after treatment, all mature bonemarrow cell populations and splenic B cell compartments were normal.

C. Discussion

The findings from this model validate the concept of tumor vasculartargeting and, in addition, demonstrate that this strategy iscomplimentary to that of direct tumor targeting. The theoreticalsuperiority of vascular targeting over the conventional approach wasestablished by comparing the in vivo antitumor effects of twoimmunotoxins, one directed against tumor endothelium, the other againstthe tumor cells themselves, in the same model. The immunotoxins wereequally potent against their respective target cells in vitro but, while100 μg of the tumor-specific immunotoxin had practically no effectagainst large solid C1300(Muγ) tumors, as little as 40 μg of theanti-tumor endothelial cell immunotoxin caused complete occlusion of thetumor vasculature and dramatic tumor regressions.

Despite causing thrombosis of all blood vessels within the tumor mass,the anti-tumor endothelial cell immunotoxin was not curative because asmall population of malignant cells at the tumor-host interface survivedand proliferated to cause the observed relapses 7-10 days aftertreatment. The proximity of these cells to intact capillaries inadjacent skin and muscle suggests that they derived nutrition from theextratumoral blood supply, but the florid vascularization and lowinterstitial pressure in those regions of the tumor rendered thesurviving cells vulnerable to killing by the anti-tumor immunotoxin(Jain, 1990; Weinstein & van Osdol, 1992), so that combination therapyproduced some complete remissions.

The time course study demonstrated that the anti-Class II immunotoxinexerted its antitumor activity via the tumor vasculature sinceendothelial cell detachment and diffuse intravascular thrombosis clearlypreceded any changes in tumor cell morphology. In contrast with theanti-tumor immunotoxin, the onset of tumor regression in animals treatedwith the anti-tumor endothelial cell immunotoxin was rapid. Massivenecrosis and tumor shrinkage were apparent in 48-72 hours afterinjection. Focal denudation of the endothelial living was evident within2-3 hours, in keeping with the fast and efficient in vivo localizationof M5/114 antibody and the endothelial cell intoxication kinetics of theimmunotoxin (t 1/10=2 hours, t 1/2=12.6 hours.

As only limited endothelial damage is required to upset the hemostaticbalance and initiate irreversible coagulation, many intratumoral vesselswere quickly thrombosed with the result that tumor necrosis began within6-8 hours of administration of the immunotoxin. This illustrates severalof the strengths of vascular targeting in that an avalanche of tumorcell death swiftly follows destruction of a minority of tumor vascularendothelial cells. Thus, in contrast to conventional tumor celltargeting, anti-endothelial immunotoxins could be effective even if theyhave short serum half lives and only bind to a subset of tumorendothelial cells.

MHC Class II antigens are also expressed by B-lymphocytes, some bonemarrow cells, myeloid cells and some renal and gut epithelia in BALB/cnu/nu mice, however, therapeutic doses of anti-Class II immunotoxin didnot cause any permanent damage to these cell populations. Splenic Bcells and bone marrow myelocytes bound intravenously injected anti-ClassII antibody but early bone marrow progenitors do not express Class IIantigens and mature bone marrow subsets and splenic B cell compartmentswere normal 3 weeks after therapy, so it is likely that any Ia⁺myelocytes and B cells killed by the immunotoxin were replaced from thestem cell pool. It is contemplated that the existence of large numbersof readily accessible B cells in the spleen prevented the anti-Class IIimmunotoxin from reaching the relatively inaccessible Ia⁺ epithelialcells but hepatic Kupffer cells were not apparently damaged byM5/114-dgA despite binding the immunotoxin. Myeloid cells are resistantto ricin A-chain immunotoxins, probably due to unique endocytic pathwaysrelated to their degradative physiologic function (Engert et al., 1991).

No severe vascular-mediated toxicity was seen in the studies reportedhere because mice were maintained on oral antibiotics which minimizedimmune activity in the small intestine.

The findings described in this example demonstrate the therapeuticpotential of the vascular targeting strategy against large solid tumors.As animal models for cancer treatment are widely accepted in thescientific community for their predictive value in regard to clinicaltreatment, the invention is also intended for use in man. Numerousdifferences between tumor blood vessels and normal ones have beendocumented (Denekamp, 1990; Jain, 1988) and are envisioned to be of usein practicing this invention. Tumor endothelial markers may be induceddirectly by tumor-derived angiogenic factors or cytokines (Ruco et al.,1990; Burrows et al., 1991) or could relate to the rapid proliferation(Denekamp & Hobson, 1982) and migration (Folkman, 1985a) of endothelialcells during neovascularization. Candidate anti-tumor endothelial cellantibodies include FB-5, against endosialin (Rettig et al., 1992), andE9 (Kumar et al., 1993) which are reportedly highly selective for tumorvascular endothelial cells. Two related antibodies developed by thepresent inventors, TEC-4 and TEC-11, against carcinoma-stimulated humanendothelial cells, show strong reactivity against vascular endothelialcells in a wide range of malignant tumors but little or no staining ofvessels in benign tumors or normal tissues. Vascular targeting istherefore envisioned to be a valuable new approach to the therapy ofdisseminated solid cancers for which there are currently no effectivetreatments.

EXAMPLE III Targeting the Vasculature of Breast Tumors

This example describes an approach for targeting the vasculature ofbreast cancer and other solid tumors in humans. This approach isexemplified through the use of bispecific antibodies to selectivelyinduce the activation antigens, Class II and ELAM-1, on the vascularendothelial cells of syngeneic breast tumors in mice and then targetingthese antigens with immunotoxins.

Murine models may first be employed. The results from such studies willbe understood to parallel the situation in humans, as mouse models arewell accepted and routinely employed for such purposes. Followingsuccessful vascular targeting in the mouse, success in man is likely ashighly specific anti-breast cancer antibodies are available (Denekamp,1984; Girling et al., 1989; Griffin et al., 1989; Lan et al., 1987;Boyer et al., 1989).

In the case of clinical (as opposed to diagnostic applications), thecentral issue is to confine the expression of the induced target antigento tumor vasculature. In the case of Class II, which is present on thevasculature of normal tissues in mice and humans (Natali et al., 1981,Daar et al., 1984; Hammerling, 1976), the objective is to suppress itsexpression throughout the vasculature and then selectively induce it ontumor vasculature. In the case of ELAM-1, which is absent from thevasculature of normal tissues (Cotran et al., 1976), the objective is toinduce its expression selectively on tumor vasculature.

A. Overview

1. Selective Induction of Class II Expression on Tumor Vasculature

C3H/He mice will be injected subcutaneously with syngeneic MM102 mammarytumor cells. The tumor cells express Ly6.2 which is a unique marker inC3H mice (Ly6.1 positive). Mice bearing solid MM102 mammary tumors willbe treated with CsA to reduce or abolish Class II expression throughoutthe vasculature. As originally shown in the dog (Groenewegen et al.,1985), and, as recently confirmed by the inventors in the mouse, CsAinhibits T cell and NK cell activation and lowers the basal levels ofIFN-γ to the extent that Class II disappears from the vasculature. Themice will then be injected with a bispecific (Fab'-Fab')anti-CD28/anti-Ly6A.2 antibody, which should localize to the tumor byvirtue of its Ly6.2-binding activity. The bispecific antibody shouldthen bind to T cells which are present in (or which subsequentlyinfiltrate (Blanchard et al., 1988) the tumor. Crosslinking of CD28antigens on the T cells by multiple molecules of bispecific antibodyattached to the tumor cells should activate the T cells via theCsA-resistant CD28 pathway (Hess et al., 1991; June et al., 1987;Bjorndahl et al., 1989). Activation of T cells should not occurelsewhere because the crosslinking of CD28 antigens which is necessaryfor activation (Thompson et al., 1989; Koulova et al., 1991) should notoccur with soluble, non-tumor cell bound, bispecific antibody. T cellswhich become activated in the tumor should release IFN-γ which shouldinduce Class II antigens on the tumor vascular endothelium (Collins etal., 1984; Pober et al., 1983) and probably on the tumor cellsthemselves (Boyer et al., 1989). Animals will then be treated withanti-Class II immunotoxins to destroy the tumor blood supply.

2. Induction of ELAM-1 Expression on Tumor Vasculature

Mice bearing solid MM102 mammary tumors will be injected with bispecific(Fab'-Fab') anti-CD14/anti-Ly6A.2 antibody. The antibody should localizein the tumor by virtue of its Ly6.2-binding activity. It should thenactivate monocytes and macrophages in the tumor by crosslinking theirCD14 antigens (Schutt et al., 1988; Chen et al., 1990). The activatedmonocytes/macrophages should have tumoricidal activity (Palleroni etal., 1991) and release IL-1 and TNF which should rapidly induce ELAM-1antigens on the tumor vascular endothelial cells (Bevilacqua et al.,1987; Pober et al., 1991). A monoclonal antibody to mouse ELAM-1 will begenerated and used as an immunotoxin to destroy the tumor blood supply.

B. Study Design and Methods

1. Suppression of Class II Expression

a) Mouse mammary tumors, MM102 and MM48

The tumors preferred for use are the mouse mammary (MM) tumors whichhave been extensively characterized by Dr. Reiko Irie and colleagues(Irie, 1971; Irie et al., 1970). The inventors have obtained from Dr.Irie (UCLA School of Medicine, CA) two transplantable tumors, MM102 andMM48. The MM102 line derives from a spontaneous mammary tumor whichoriginated in a C3H/He mouse. The MM102 tumor carries an antigen whichis closely related to, or identical to, Ly6A.2 (Seto, et al., 1982).Since the C3H/He mouse expresses Ly6.1 and not Ly6.2, this marker istumor-specific in syngeneic mice. The MM48 tumor, a variant of theoriginal tumor, lacks Ly6A.2 and provides a specificity control in theproposed studies. Both tumors form continuously growing solid tumorswhen injected into the subcutaneous site.

b) Monoclonal antibodies

For targeting the Ly6A.2 antigen, the inventors have obtained theanti-Ly6A.2 hybridoma, S8.106, from Dr. Ulrich Hammerling (MemorialSloan-Kettering Cancer Center, N.Y.). This hybridoma secretes a mouseIgG_(2a) antibody (Kimura, et al., 1980) which has been shown to reactspecifically with MM102 and other Ly6A.2 expressing MM tumors (Seto, etal., 1982).

An appropriate anti-mouse CD28 antibody (Gross, et al., 1990) is thatobtainable from Dr. James Allison (University of California, Calif.).Ascitic fluid from hybridoma-bearing animals is also available forsynthesizing the bispecific antibody. The antibody is a hamster IgG.

Isotype-matched negative control antibodies will be the WT1 antibody(anti-human CD7) which is a mouse IgG₂ a and a hamster IgG of irrelevantspecificity from the ATCC.

Antibodies will be purified on staphylococcal Protein A coupled toSepharose, or by ion exchange and size exclusion chromatography onSepharose 4B as described by Ghetie, et al. (1988). The ability of thepurified anti-Ly6A.2 antibody to bind to MM102 cells and of theanti-CD28 antibody to bind mouse T cells will be confirmed by FACSanalyses as described by Burrows et al., (1991).

Purified antibodies will be filtered through 0.22 μm membranes,aliquotted, and stored at -70° C.

c) Preparation of Fab' fragments

F(ab')₂ fragments of purified anti-Ly6A.2 and anti-CD28 antibodies willbe prepared by pepsin digestion, as described by Glennie et al. (1987).Purified antibodies (5-10 mg) will be dialyzed against 0.1M sodiumacetate, pH 4.1, and digested with 4% (w/w) pepsin at 37° C. Thedigestion will be followed by SDS- PAGE and terminated by raising the pHwhen optimal digestion is achieved. Undigested IgG will be removed bychromatography on a Sephacryl S-200 column equilibrated with PBS. TheF(ab')₂ fragments will be analyzed by SDS-PAGE and, if detectable levelsof undigested antibody should remain, the F(ab')₂ fragments will befurther purified by removal of undigested antibody on a ProteinA-Sepharose column. Fab' fragments will be prepared from F(ab')₂fragments by reduction with 5 mM DTT for 1 hr at 25° C. , followed byremoval of free DTT on a Sephadex G-25 column equilibrated againstphosphate-EDTA buffer (Glennie et al., 1987)

d) Preparation of anti-Ly6A.2/anti-CD28 bispecific antibodies

For the production of anti-Ly6A.2-anti-CD28 bispecific antibodies, Fab'fragments of each antibody will be initially prepared as above and willbe left unalkylated. Heterodimer molecules will be prepared as describedby Glennie et al. (1987). Fab' fragments will be reduced with DTT in0.2M Tris-HCl buffer, pH 8.0, containing 10 Mm EDTA for 60 min. at roomtemperature. One of the Fab' fragments will be then reacted withEllman's reagent (2 mM) for 1 hour at room temperature in acetatebuffer, pH 5.0. The free Ellman's reagent will be separated using aSephadex G-25 column. The derivatized Fab' fragment will be then mixedwith the other reduced Fab' and allowed to react at room temperature for24 hours. Bispecific antibodies will be separated from remaining Fab'fragments by gel filtration over Sephacryl S-200 columns.

e) Confirmation of cell binding-capacity of anti-Ly6A.2/anti-CD28bispecific-antibody

FACS analyses will be performed to verify the dual cell-binding capacityof the bispecific antibody. MM102 tumor cells (grown as an ascites) willbe treated for 30 minutes at 4° C. with the bispecific antibody (10μg/10⁶ cells) and washed. The tumor cells will then be incubated withfluoresceinated goat anti-hamster immunoglobulin for 30 minutes at 4° C.and washed again. The fluorescence associated with the cells will thenbe measured using the FACS. Positive staining of tumor cells coated withbispecific antibody and lack of staining of cells coated withanti-Ly6A.2 antibody alone will confirm that the bispecific antibody isintact and is capable of binding tumor cells. The study will be repeatedusing a CD28 positive mouse T cell lymphoma line (e.g., EL4) and withfluoresceinated goat anti-mouse immunoglobulin as the detecting antibodyto confirm that the bispecific antibody has CD28-binding capacity.

f) Activation of T cells by anti-Ly6A.2/anti-CD28 bispecific antibodyplus MM102 tumor cells

It will be important to confirm that tumor cells coated with thebispecific antibody, but not free bispecific antibody, are able toactivate T cells in a CsA-resistant fashion. T cells will be enrichedfrom the spleens of C3H/He mice by depleting B-cells and macrophagesaccording to the procedure of Lee and colleagues 1990 (Lee, et al.,1990). Spleen cells are treated with mouse anti-Class II antibody andthe Class II-expressing cells are removed by treating them with goatanti-mouse IgG-coupled magnetic beads and withdrawing them with a strongmagnet. The non-adherent cells are decanted and are treated further toremove residual B cells and macrophages by successive rounds oftreatment with anti-J11D plus BRC and anti-MAC-1 antibody plus goatanti-rat serum. After these procedures, the remaining cells are ≧95% Tcells and <3% Ig positive.

T cells will be cultured (0.5 to 1×10⁵ cells/0.2 ml) in medium in thewells of 96-well plates. Various concentrations of anti-CD28 IgG,anti-CD28 Fab' or anti-Ly6A.2/anti-CD28 bispecific antibody will beadded together with various concentrations of one of the followingcostimulants: PMA, IL1 or anti-CD3 IgG. CsA (0.5 μg/ml) will be added toan identical set of cultures. The cultures will be incubated at 37° C.for 3 days, ³ H-thymidine (1 μCi/culture) will be added and the platesharvested 24 hours later. These studies should confirm that bivalentanti-CD28, but not monovalent Fab' anti-CD28 or the bispecific antibody,stimulate T cells and that the stimulation is not CsA inhibitable.

Next, MM102 and MM48 cells, obtained from ascitic tumors of C3H/He mice,will be treated with mitomycin C (25 μg/ml) for 20 minutes at 37° C. Thecells will then be washed and the above study repeated with theinclusion of 0.5 to 1×10⁵ mitomycin- treated MM102 or MM48 cells alongwith the T cells in the cultures. The MM102 cells, but not the MM48cells, should present the bispecific antibody to the T cells and,together with the costimulant, induce their stimulation.

g) Confirmation that injection of anti-Ly6A.2/anti-CD28 bispecificantibody into CsA-treated MM102 tumor-bearing mice results in inductionof Class II selectively on tumor vasculature

C3H/He mice will be injected subcutaneously with 10⁶ MM102 or MM48 tumorcells. One day later they will start daily treatments with CsA (60mg/kg/day) given either orally dissolved in olive oil or injectedintraperitoneally. After 10-14 days, when the tumors will have reached1.0-1.3 cm in diameter, and when Class II will have disappeared from thevasculature, mice will be injected with 50-100 μg ofanti-Ly6A.2/anti-CD28 bispecific antibody. Other mice will receivevarious control treatments, including unconjugated anti-Ly6A.2 oranti-CD28 (Fab' and IgG) or diluent alone. Two or three days later, themice will be sacrificed and the tumors and various normal tissues willbe removed for immunohistochemical examination. Frozen sections will becut and stained for the presence of Class II antigens and for thepresence of hamster immunoglobulin using indirect immunoperoxidasetechniques, as presented in the foregoing examples.

Upon demonstration that Class II antigens are strongly and selectivelyexpressed on the vasculature of MM102 tumors but not on MM48 tumors, thetumor therapy studies below will be carried out. If Class II antigensare absent from tumor vasculature but hamster immunoglobulin is present,this would indicate that the bispecific antibody had localized to thetumor, as anticipated from prior studies with analogous bispecificantibodies (Perez, et al., 1985; Garrido, et al., 1990), but that T cellactivation had not occurred sufficiently for IFN-γ secretion to ensue.If so, the presence of T cells will be verified by staining frozen tumorsections with anti-CD28 and anti-CD3 antibodies. If T cells are present,again as would be anticipated from prior studies (Koulova, et al., 1991;Perez, et al., 1985), the failure to get Class II induction might beattributable to the need for two signals for T cell activation, i.e. a2nd signal might be missing. This will be checked by coadministering ananti-Ly6A.2/anti-CD3 bispecific antibody, which together with theanti-Ly6A.2/anti-CD28 bispecific, should provide the signalling neededfor T cell activation.

h) Synthesis of anti-class II-SMPT-dgA immunotoxin

Immunotoxins directed against murine class II MHC molecules will beprepared by linking the rat monoclonal anti-murine I-A^(k) antibody todeglycosylated ricin A (dgA) using the disulfide cleavable crosslinker,SMPT (Thorpe, et al., 1988). Affinity purified antibody molecules willbe derivatized by reaction with a five-fold molar excess of SMPT inborate buffer, pH 9.0, for 60 min. at room temperature. Free SMPT willbe removed by passage through a Sephadex G-25 column equilibratedagainst phosphate buffered saline containing EDTA (1 mM, PBSE). Underthese conditions, an average of 1.7 molecules of SMPT are introduced perimmunoglobulin molecule. Next, the derivatized antibody will be allowedto react with reduced dgA for 72 hrs. at room temperature. Under theseconditions, immunoconjugates form through formation of disulfide linkagebetween sulfhydryl groups on dgA molecules and SMPT. Immunoconjugateswill be separated from free dgA by gel filtration in Sephacryl S-200columns and from unreacted antibody by passage through Blue-Sepharoseand elution with phosphate buffer containing 0.5M NaCl. Purity ofimmunoconjugates will be assessed by SDS-PAGE.

i) Tumor therapy studies

C3H/He mice will be injected subcutaneously with 10⁶ MM102 or MM48 tumorcells and, one day later, will start daily treatments with CsA (60mg/kg/d). When the tumors have grown to 1.0-1.3 cm diameter, the micewill receive an intravenous injection of 50-100 μg anti-Ly6A.2/anti-CD28bispecific antibody (perhaps together with anti-Ly6A.2/anti-CD3bispecific antibody if indicated by the studies in Section (h) above).Two or three days later, 100 μg of the anti-Class II immunotoxin will beadministered intravenously. Anti-tumor effects will be monitored bymeasuring the size of the tumors at regular intervals and byhistological examination as in Section C. The specificity of anyanti-tumor effects will be established by comparing the anti-tumoreffects with those in mice which receive various control treatments,including unconjugated anti-Ly6A.2 Fab' and IgG, unconjugated anti-CD28Fab' and IgG, and anti-Class II immunotoxin alone.

2. Induction of ELAM-1 on tumor vasculature by anti-Ly6A.2/anti-CD14bispecific antibody

a) Raising of anti-ELAM-1 monoclonal antibodies

i) Induction of ELAM-1 on SVEC cells for immunization

Expression of cytokine-induced adhesion molecules on SVEC murineendothelial cells will be induced by stimulation of SVEC cell monolayerswith a cocktail of rMuIL-1β (50 I.U./ml), rMuTNFα (100 IU/ml) andbacterial endotoxin (100 ng/ml) for 4 hrs at 37° C. , as described forthe induction of human ELAM-1 (Bevilacqua, et al., 1987). Preliminaryevidence suggests that SVEC cells activated in this manner expressmurine ELAM-1, since radiolabeled U937 cells, which bear the ELAM-1agent, display increased adhesion to activated SVEC cells within 2 hrsof the addition of the cytokines. The increased endothelial celladhesiveness peaked at 4-6 hrs., as previously reported for humanELAM-1. Although cytokine-activated SVEC cells also displayed long-term(up to 48 hrs.) adhesiveness to U937 cells, this was probably not due toICAM-1/LFA-1 or VCAM-1/VLA-4 interactions, since the assays were carriedout at 4° C. , under shear stress conditions, which inhibits anyadhesive interactions other than those between selections and theircarbohydrate agents (Spertini, et al., 1991). Increased adhesiveness atthe later time points was probably mediated by the selection LAM-1(Mel-14) on U937 cells and its agent (the MECA 79 antigen) on SVEC cells(81). In subsequent studies, this pathway will be blocked by theinclusion of Mel-14 and/or MECA 79-specific antibodies in the adhesionassays (Imai, et al., 1991).

ii) Immunization

Rat monoclonal antibodies will be raised against inducible proteins onmouse endothelial cells. SVEC cells will be stimulated for 6 hrs., aspreviously described, before immunization of Wistar rats. The rats willbe boosted three weeks following the initial injection withidentically-prepared SVEC cells. Serum from the injected rats will betested for the presence of antibodies specific for induced proteins onendothelial cells 7-10 days after the second boost, using FACS analysisof induced and non-induced SVEC cells. Additional boosting and screeningwill be repeated as necessary.

Once antibody levels have been detected in acceptable titers, rats willbe given a final boost with induced SVEC cells and their spleens removedafter 3 days. Splenocytes will be fused with Y3 μg1.2.3 rat myelomacells according to standard protocols using poly-ethylene glycol 4000(82). Hybridomas will be selected using HAT medium and the supernatantsscreened using FACS analysis.

iii) Screening

Those hybridomas secreting antibodies reacting with cytokine-induced butnot with resting SVEC cells will be selected for furthercharacterization. Reactivity will be assessed by indirectimmunofluorescence with hybridoma supernatants and FITC-labeled mouseanti-rat immunoglobulin antibodies. The selected hybridomas will beexpanded, and the immunoglobulin purified from their culturesupernatants. Confirmation of ELAM-1 reactivity will be carried out asdescribed below.

iv) Characterization of antigens

The physicochemical properties of the precipitated antigens will beinvestigated after activating SVEC cells with cytokines in the presenceof cycloheximide or tunicamycin and by immunoprecipitation ofantibody-reactive molecules from lysates of ³⁵ S-methionine-labeled SVECcells, using the selected MAbs. Immunoprecipitates will be subsequentlyanalyzed by SDS-PAGE. Confirmation of murine ELAM-1 reactivity will becarried out by comparison of the precipitated material and human ELAM-1using SDS-PAGE, one-dimensional proteolytic maps with staphylococcal V8protease and NH₂ -terminal sequences.

b) Preparation of anti-Ly6A.2-anti-CD14 bispecific antibodies

Bispecific antibodies will be constructed using Fab' fragments derivedfrom anti-Ly6A.2 and anti-CD14 monoclonal antibodies, essentially asdescribed in the previous section. Several anti-mouse CD14 monoclonalantibodies have been raised (23). We feel it is premature to approachthese workers with a view to establishing a collaboration until we haveraised anti-mouse ELAM-1 monoclonals and verified their performance asimmunotoxins.

c) Synthesis and cytotoxicity testing of anti-ELAM-1 inmunotoxins

Immunotoxins directed against murine ELAM-1 will be constructed bycross-linking monoclonal anti-mouse ELAM-1 antibodies (as characterizedabove) to dgA using SMPT. The procedure involved will be identical tothat described in the previous sections. Activity will be assessed bytoxicity studies with cytokine-activated SVEC cells.

d) Confirmation of ELAM-1 induction on tumor vasculature and not onnormal vasculature

C3H/He mice bearing 1.0-1.3 cm MM102 or MM48 tumors will be injected i.vwith anti-Ly6A.2/anti-CD14 bispecific antibody or with various controlmaterials including unconjugated anti-Ly6A.2 and anti-CD14 antibodies(Fab' and IgG) and diluent alone. Tumors will be removed at varioustimes and cryostat sections will be cut and stained with rat monoclonalantibodies to murine ELAM-1, using standard indirect immunoperoxidasetechniques. The presence of the bispecific antibody on tumor cells willbe verified by staining for rat immunoglobulin. Resident macrophages andinfiltrating monocytes will be detected by indirect immunoperoxidasestaining with anti-Mac-1 (CD 11b/CD 18) antibodies. Cytokine-producingcells will be identified in serial cryostat sections of tumors by insitu hybridization with ³⁵ S-labeled antisense asymmetric RNA probes formurine IL-1β and TNFα mRNA.

e) Tumor therapy studies

C3H/He mice bearing 1.0-1.3 cm MM102 or MM48 tumors will be injectedwith 50-100 μg anti-Ly6A.2/anti-CD14 bispecific antibody or with variouscontrol materials including unconjugated anti-Ly6A.2 and anti-CD14antibodies (Fab' and IgG) and diluent alone. One to three days later,the mice will receive intravenous injections of anti-ELAM-1 immunotoxin,an isotype-matched immunotoxin of irrelevant specificity or unconjugatedanti-ELAM-1 antibody. Anti-tumor effects will be monitored by measuringthe size of the tumors at regular intervals and by histologicalexamination, as in the preceding examples.

Mice will be injected subcutaneously with 10⁶ MM102 or MM48 tumor cells(in 0.1 ml saline) either on the abdominal wall or on the flank. In somestudies, cyclosporin A (60 mg/kg/day) will be injected intraperitoneallyor given in the drinking water. The mice will be observed dailythereafter and the dimensions of the tumor will be measured. When thetumor reaches a diameter of 1.0-1.3 cm, the mice will receive aninjection of bispecific antibody (0.1 ml in saline) into a tail vein andthen 2-3 days later will receive an intravenous injection ofimmunotoxin, again into the tail vein. The study is terminated byeuthanizing the mice when their tumors reach 1.5-2 cm in diameter in anydimension.

Each group will comprise 8-10 animals, and there will generally be 5 or6 treatment groups making a total of 40-60 mice per study. One suchstudy will be performed per month.

f) Raising monoclonal antibodies

Adult Wistar rats will be used. Rats will be immunized by injecting themi.m with mouse endothelial cells (SVECs) homogenized in 0.1 ml of a50:50 mixture of Freund's incomplete adjuvant and saline. Rats will beboosted 1 month and 2 months later in the same manner. 7-10 days afterthe second boost, 0.1 ml blood will be removed from tail vein and theserum will be analyzed for the presence of antibody. If sufficientlypositive, the rats will be given a final i.m boost with SVEC cells and 3days later, the rats will be euthanized their spleens dissected out formonoclonal antibody production.

g) Raising ascites

BALB/c nude mice will be injected intraperitoneally with 0.5 ml Pristane(2, 6, 10, 14-tetramethylpentadecane) 2 to 4 weeks before being injectedintraperitoneally with rat hybridoma cells. The mice will be weigheddaily and euthanized when their body weight increases by 20% or more dueto hybridoma growth in the peritoneal cavity. The contents of theperitoneal cavity will then be drained and monoclonal antibodiespurified from the ascitic fluid.

h) Choice of species and number of animals

Mice: The antitumor effects of immunotoxins in animals cannot bepredicted from tissue culture studies. Such factors as hepaticentrapment, blood clearance rates and binding to serum components makesit essential that intact animal systems are used for evaluation. Thechoice of mice as the test animal is determined by the fact that inbredstrains exist in which mammary tumors will grow reproducibly. The numberof animals (8-10) per treatment group is the minimum for statisticallysignificant differences between different treatment groups to becomeapparent. The number of treatment groups (5-6) per study is the minimumfor an effect of a specific immunotoxin to be distinguished from aneffect of its components (antibody alone, ricin A chain alone, ormixtures of the two) and for superiority over control immunotoxin ofirrelevant specificity to be demonstrated.

Rats: Since antibodies are to be raised to mouse endothelial cellantigens, it is best to use another species for immunization. Rats arepreferred for these studies because they are inbred and respondconsistently to the immunogen.

EXAMPLE IV Identification and Characterization of the Tumor EndothelialCell Marker, Endoglin, and Antibodies thereto

This example describes the generation of two new monoclonal antibodies,TEC-4 and TEC-11, directed against a tumor vasculature antigen. TEC-4and TEC-11 are shown to recognize endoglin, which is shown to beassociated with growth and proliferation of human tumor endothelialcells in vitro and in vivo. Endoglin is selectively upregulated onvascular endothelial cells in a broad range of malignant tumors and isenvisioned to provide a suitable marker for use in the diagnosis andtherapy of miscellaneous solid tumors.

A. Materials and Methods

1. Cells and culture conditions

Cell lines were obtained from the Imperial Cancer Research Fund, London(U.K.) Tissue Bank unless otherwise indicated. SP2/0 murine myelomacells, SAOS osteosarcoma cells and the ECV-304 human endothelial cellline were obtained from the American Type Culture Collection, Rockville,Md. A375M and T8 human melanoma lines were obtained from Dr. I. R. Hart(Imperial Cancer Research Fund, London, U.K.). NCI-H146 and SCC-5 humanlung cancer cell lines were obtained from Dr. J. D. Minna (U.T.Southwestern Medical Center, Dallas, Tex.). L428 and L540 humanHodgkins/Sternberg-Reed cells were obtained from Dr. A. Engert(Department of Medicine, Cologne, Germany).

The murine endothelial cell lines used were SV40-transformed mouse lymphnode endothelial cells (SVEC, O'Connell et al., 1990), obtained from Dr.M. Edidin (Johns-Hopkins, Baltimore, Md.); LEII, obtained from Dr. W.Risau (Max-Planck Institute, Martinsried, Germany); and mouse pulmonarycapillary endothelial cells (MPCE) obtained from Prof. A. Curtis(Department of Cell Biology, Glasgow, U.K.).

Primary endothelial cell cultures from chinese hamster epididymal fatpad and bovine heart were established using the methods described byBjorntorp et al. (Bjorntorp et al., 1983) and Revtyak et al. (Revtyak etal., 1988).

Human umbilical vein endothelial cells (HUVEC) were isolated from freshtissue by the method of Jaffe et al. (Jaffe et al., 1973) or werepurchased from Clonetics Corp., San Diego, Calif. Cultures weremaintained in gelatin-coated flasks in Medium 199 (Gibco-Biocult, Ltd.,Paisley, U.K.) supplemented with Earle's salts, 200% (v/v) fetal calfserum, endothelial cell growth supplement (ECGS, 0.12 mg/ml), 0.09 mg/mlheparin, glutamine and antibiotics at 37° C. in 5% CO₂ in air or inEndothelial Growth Medium (Clonetics) at 37° C. in 100 CO₂ in air. Allother cell lines were maintained in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% (v/v) fetal calf serum, 2.4 mM L-glutamine,200 units/ml penicillin, 10 μg/ml streptomycin, 100 μM non-essentialamino acids, 1 μM Na Pyruvate and 18 μM HEPES at 37° C. in 10% CO₂ inair.

Murine L-cell transfectants expressing human endoglin were produced asdescribed by Bellon et al. (1993).

2. Antibodies

The F8/86 mouse IgG1 anti-human von-Willebrands Factor antibody (Naieumet al., 1982) was purchased from DAKO Ltd (High Wycombe, U.K.) and usedat 1:50 dilution. A mouse IgG1 anti-human vitronectin receptor antibody,LM142 (Cheresh, 1987) was obtained from by Dr. D. Cheresh (ScrippsClinic, La Jolla, Calif.). The mouse IgG1 anti-rat Thy 1.2 antibody(Mason and Williams, 1980) was used as an isotype matched control forF8/86 and LM142. The mouse myeloma proteins TEPC-183 (IgM_(k)) andantibody MTSA (IgM_(k)), which do not react with human tissues, wereused as negative controls for TEC-4 and TEC-11. The mouse IgG1anti-endoglin antibody 44G4 (Gougos and Letarte, 1988) has beendescribed previously.

3. Immunoprecipitation

HUVEC were metabolically labelled by overnight incubation in 0.1 mCi/10⁶cells ³⁵ S-methionine in methionine-free RPMI supplemented with 20%(v/v) fetal calf serum, ECGS, glutamine heparin and antibiotics asdescribed above. Monolayers were washed twice in PBS and adherent cellswere lysed in NET buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mMEDTA, 0.5% NP-40) supplemented with 0.1M iodoacetamide to inhibitdisulfide exchange.

For immunoprecipitation, preformed immunocomplexes were used asdescribed by Li et al. (1989). 0.2 mg of purified MAb in 200 μl of NETbuffer was mixed with 250 μl of GAMIg (goat anti-mouse immunoglobulin)serum and incubated for 30 min. at room temperature. Immunocomplexeswere pelleted by centrifugation and the pellet was washed two times in300 μl of NET buffer. One ml of lysate from 5×10⁷ ¹²⁵ I-labeled cellswas mixed with immunocomplexes (prepared as above) on a rotator at 4° C.for 1 hr. The lysate containing the immunocomplexes was layered on topof a discontinuous sucrose gradient consisting of 750 μl each of 40(bottom), 30, 20, and 10% (top) sucrose in 12×75-mm tubes. Tubes werecentrifuged at 600 g for 20 min. at room temperature. The sucrose wasdecanted and the pellets were resuspended in a small amount of PBS.Samples were transferred to new tubes and then centrifuged. The pelletwas boiled for 1 min. in sample buffer with or without 2-mercaptoethanol(2-ME) and electrophoresed on 12.5% slab gels for 16 hr at 15 mA. Thegels were dried and exposed to film for 24 hr at -70° C.

4. Complement fixation

The ability of TEC-4 and TEC-11 to fix complement was assessed asfollows. Primary antibody incubations and washing were performed as forindirect immunofluorescence. After the third wash, HUVEC wereresuspended in 50 μl of a 1/10 dilution of guinea pig complement (ICN,High Wyecombe, U.K.) for 20 min. at 37° C. , at which point 50 μl of0.25% (w/v) trypan blue was added and cell number and plasma membraneintegrity were estimated visually.

5. Indirect Immunofluorescence

Tumor cells and endothelial cells were prepared for FACS analyses asdescribed by Burrows et al. (1991). All manipulations were carried outat room temperature. 50 μl of cell suspension at 2-3×10⁶ cells/ml inPBS-BSA-N₃ were added to the wells of round-bottomed 96 well microtiterplates (Falcon 3910). Primary antibodies (10 μg/ml) were added in 50 μlvolumes, and the plates sealed. After 15 min., the cells were washed 3times by centrifuging the plates at 800×g for 30 s, removing thesupernatants by flicking and patting dry on absorbent tissue, andresuspending the cells in 150 μl/well PBS-BSA-N₃. Fluoresceinisothiocyanate (FITC)-conjugated rabbit antibodies against mouseimmunoglobulins (DAKO Corp., Carpinteria, Calif.), diluted 1:20 inPBS-BSA-N₃. were distributed in 50 μl volumes into the wells. The cellswere incubated for a further 15 min. and washed as before.Cell-associated fluorescence was measured on a FACScan(Becton-Dickenson, Fullerton, Calif.). Data were analyzed using theLYSYSII program. The mean fluorescence intensity (MFI) of cells treatedwith the control antibody, MTSA, was subtracted from that of cellstreated with TEC-4 or TEC-11 to obtain the specific MFI attributable toantigen binding.

For competitive binding inhibition assays, biotinylated TEC-4 or TEC-11antibodies were mixed with unlabelled TEC-4, TEC-11 or controlantibodies at ratios of 1:1, 1:10 and 1:100. Indirect immunofluorescentstaining of HUVEC was carried as before except that biotinylatedantibodies bound to the cells were detected with a 1:50 dilution ofstreptavidin-phycoerythrin (Tago, Inc., Burlingame, Calif.). Percentblocking of biotinylated antibodies was calculated as follows:

    % blocking=MFI in presence of blocking Ab (background MFI)/MFI in presence of non-specific Ab (background MFI)

Indirect immunofluorescence of L-endoglin transfectants was carried outas described by Bellon et al. (1993). Parental L cells and L celltransfectants expressing human endoglin (1×10⁶ cells) were incubated for45 min. at 4° C. with MAb 44G4 IgG (10 ug/ml), TEC-4 (10 μg/ml) orTEC-11 (10 μg/ml). Cells were then washed and incubated withFITC-conjugated F(ab')₂ goat anti-mouse IgG (H+L)(Tago).

6. Cellular protein and nucleic acid analyses

HUVEC were stained with TEC-11 at 20 μg/ml as described in `IndirectImmunofluorescence`, and sorted into TEC-11^(lo) and TEC-11^(hi)populations using a FACStar Plus cell sorter.

Total cellular protein content was estimated from the uptake of freeFITC as described by Stout and Suttles (1992). Sorted cells werecentrifuged at 150×g, 5 min., and fixed in cold 70% ethanol for 15 min.The cells were pelleted as before, resuspended in PBS containing 20μg/ml FITC and incubated on ice for 60 min. before being washed andanalyzed on the FACStar Plus. The ethanol fixation effectively removedany prior fluorescent label as evidenced by the lack of detectablefluorescence of controls which were not incubated with FITC.

Acridine orange staining of DNA and RNA was performed according to amethod adapted from that of Darzynkiewicz et al. (Darzynkiewicz et al.,1976). Sorted cells were centrifuged at 150×g for 5 min. and resuspendedat 1.5×10⁶ cells/ml in 200 μl of DMEM/10% FCS. 0.5 ml 0.15Mcitrate/phosphate buffer, pH 3.0, containing 0.1% (v/v) Triton-X-100(Sigma), 0.2M sucrose and 0.1 mM EDTA was added and the cells weremaintained at 4° C. Immediately before analysis, 0.5 ml 0.15Mcitrate/phosphate buffer, pH 3.8, containing 1M NaCl and 0.002% (w/v)acridine orange (Polysciences Inc., Warrington, Pa.) was added. After 5min. at room temperature, the fluorescence intensities of individualcells were measured in the FACStar Plus. Free nuclei and cell doubletswere excluded from the data collection. Green and red fluorescence wereplotted against each other in a 2-dimensional dot plot that enablescells to be assigned to different stages of the cell cycle (G₀, G₁, S,G₂ + M) according to relative DNA and RNA content (Darzynikiewicz etal., 1976).

7. Immunohistochemistry

Human and animal tissue samples were snap-frozen over liquid nitrogen,mounted in OCT Compound (Miles, Inc., Elkhart, Ind.) and 8 μM sectionswere cut in a Tissuetek 2 cryostat (Baxter) onto slides precoated with3-aminopropyltriethoxysilane (Sigma). Sections were stored at -80° C.until required. Indirect streptavidin-biotin immunoperoxidase stainingwas carried out as follows. Sections were air-dried at room temperaturefor 30 min., fixed in acetone for 15 min., rehydrated in PBS for 5 min.and incubated in a humidified chamber for 45-60 min. with purifiedprimary antibodies at 10 μg/ml in PBS-0.2% BSA or with undiluted culturesupernatant. After 2 washes in PBS, the sections were incubated for30-45 min. with biotinylated F(ab')₂ sheep anti-mouse IgG (H+L) (Sigma#B-6774) diluted 1:200 in PBS-0.20% BSA. After a further 2 washes inPBS, the sections were incubated for 30-45 min. withstreptavidin-biotin-horseradish peroxidase complex (Strept ABC Complex,DAKO #K377) diluted 1:100 in PBS-0.2% BSA. After a final 2 washes, thereaction product was developed using 0.5 mg/ml 3',3'-diaminobenzidine(DAB, Sigma) or 1 mM 3-amino-9-ethylcarbazole (AEC, Sigma) containing0.01% (v/v) hydrogen peroxide. The sections were counterstained withMayer's hematoxylin (Sigma) for 10-30 s, washed in tap water and mountedin CrystalMount (Biomeda Corp., Foster City, Calif.).

B. Results

1. Production of TEC-4 and TEC-11 antibodies

The TEC4 and TEC11 monoclonal antibodies were raised and selectivelyscreened in the following manner. HT-29 human colonic adenocarcinomacells were obtained from the Imperial Cancer Research Fund CentralTissue Bank and seeded at 25% of confluent density in tissue cultureflasks in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10%v/v fetal calf serum (FCS), 2.4 mM L-glutamine, 200 units/ml penicillinand 100 μg/ml streptomycin. The cells were allowed to grow to fullconfluence over 4-5 days incubation at 37° C. in a humidified atmosphereof 90% air/10% CO₂ before the supernatant tissue culture medium(hereafter referred to as HT-29 tumor-conditioned medium, HT-29 TCM) wasremoved, filtered through a 0.22 μM filter to ensure sterility and toremove any particulate matter, and stored at 4° C. for no more than oneweek before use. HT-29 human adenocarcinoma cells were used to prepareTCM because they had previously been shown to secrete angiogenic intotheir culture medium (Rybak et al., 1987) and angiogenic has been foundto induce neovascularization (i.e., profound alteration of endothelialcell behavior) in an in vivo assay (Fett et al., 1987).

Human umbilical vein endothelial cells (HUVEC) were incubated in Medium199 supplemented with 20% w/v FCS, glutamine and antibiotics and mixed1:1 with HT-29 TCM. After 48-72 hours at 37° C. the endothelial cellswere harvested non-enzymatically, using a rubber policeman, and 1-2×10⁶cells were injected intraperitoneally into a BALB/c mouse. This entireprocedure was repeated three times at two to three week intervals, thefinal injection being by the intravenous route.

Three days later, splenocytes from the immunized animal were fused withSP2/O murine myeloma cells at a ratio of 1:2 using PEG2000 (Kohler andMilstein, 1975; Tazzari et al., 1987). The cell mixture was introducedinto the wells of 96-well flat bottomed microtiter plates along with3×10⁴ syngeneic peritoneal feeder cells per well. Twenty-four hourslater 100 μl of medium containing hypoxanthine, ammopterin and thymidine(HAT Medium) was added to select for fused cells (hybridomas). Thecultures were fed with additional HAT Medium at 3 day intervals.

When hybridomas had grown to high density, 50 μl samples of supernatantwere taken and screened by galactosidase anti-galactosidase (GAG) ELISA(Burrows et al., 1991) for antibodies reactive with HT-29-activatedHUVEC. All positive wells were seeded into 24-well plates, expanded byfurther culture in HAT medium, and retested 7-10 days later by the sametechnique. Antibodies that bound to HUVEC in the ELISA were furthertested for reactivity with HUVEC cell surface determinants by FACS (see`Indirect Immunofluorescence`). All positive wells were harvested andsamples stored in liquid nitrogen. The remaining cells from eachpositive well were cloned in 96 well plates by the limiting dilutionmethod (Kohler and Milstein, 1975).

When the clones had grown to high density, 50 μl samples of supernatantwere taken and assayed by GAG ELISA against HT-29 TCM-activated HUVECand `resting` HUVEC grown in the absence of tumor-derived factors. Anywells which showed significantly greater reactivity with HT-29TCM-activated HUVEC than with control HUVEC were recloned and expandedto culture flasks to provide adequate supernatant for subsequentlyscreening for lack of reactivity with quiescent HUVEC in frozen sectionsof human umbilical vein (see `Immunohistochemistry`).

Supernatants from these expanded clones were screened by standardindirect immunoperoxidase techniques (Billington and Burrows, 1987) bysequential incubation with F(ab)₂ sheep anti-mouse IgG (1:200, Sigma)and streptavidin-biotin-horseradish peroxidase complex (1:100,Dakopatts) against a small panel of normal and malignant human tissueson cryostat sections. After this round of screening two clones, 1G4(TEC4) and 2Gll (TEC11), were selected for further study on the basis ofsignificantly greater reactivity with endothelial cells in sections ofsolid tumors than with those in sections of normal tissues.

Both TEC4 and TEC11 antibodies were isotyped as IgM using theImmunotype™ mouse monoclonal antibody isotyping kit (Sigma Chemical Co.,St. Louis, Mo.). TEC-4 and TEC-11 were purified from tissue culturesupernatant by ammonium sulfate precipitation followed by SephacrylS-300 size-exclusion chromatography and affinity chromatography on aMannose-Binding Protein column (Pierce Chemical Co., Rockford, Ill.).TEC-4 and TEC-11 antibodies were biotinylated by incubation with a40-fold molar excess of N-hydroxy-succinimidobiotin amidocaproate(Sigma) for 1 h at room temperature followed by dialysis against 2changes of PBS.

2. Evidence that TEC-4 and TEC-11 recognize Endoglin

TEC-4 and TEC-11 immunoprecipitated a molecule that migrated as a 95 kDaspecies when analyzed on SDS-polyacrylamide gels under reducingconditions (FIG. 13(a), lanes 2-4). When isolated and analyzed undernon-reducing conditions, the molecule moved predominantly as a 180 kDahomodimer (FIG. 13(a), lanes 5-7). These biochemical characteristicswere consistent with those described previously for endoglin; indirectimmunofluorescence analysis of the binding of TEC-4 and TEC-11 to murineL-cells transfected with human endoglin was therefore performed todetermine whether the antibodies indeed recognize endoglin. As shown inFIG. 13(b), TEC-4, TEC-11 and the reference anti-endoglin antibody,44G4, all reacted strongly with endoglin-transfectant L-cells but wereunreactive with parental L-cells.

3. Cross-blocking of TEC-4 and TEC-11 antibodies

Binding to L-endoglin transfectants (FIG. 13) and reciprocal preclearingbefore immunoprecipitation confirmed that TEC-4 and TEC-11 react withthe same protein. Competitive inhibition of binding to HUVEC was carriedout to determine whether TEC-4 and TEC-11 recognize the same ordifferent epitopes on the endoglin molecule (FIG. 14).

TEC-4 and TEC-11 blocked themselves in a dose dependent manner, withTEC-11 being slightly more efficient that TEC-4 (FIG. 14). TEC-11partially inhibited binding of TEC-4 by 51% at a ratio of 1:1, butincreasing the ratio of TEC-4:TEC-11 to 1:10 did not increase theblocking. By contrast, TEC-4 inhibited TEC-11 binding by only 1 % at1:1, but this blocking increased in a dose-dependent fashion to reach20% at a TEC-11:TEC-4 ratio of 1:100 (FIG. 14). The blocking effectswere specific since neither TEC-4 nor TEC-11 blocked the binding of theanti-vitronectin receptor antibody LM142 to HUVEC, nor did LM142interfere with TEC-4 or TEC-11 binding.

The epitopes recognized by TEC-4 and TEC-11 were distinct from that of44G4, because 44G4 did not block TEC-4 or TEC-11, even at a ratio of100:1.

Taken together, the above results indicate that (i) TEC-4 and TEC-11recognize distinct but spatially close epitopes on the endoglinmolecule, because neither antibody inhibited the binding of the other asefficiently or as completely as it blocked itself, and (ii) TEC-11antibody has a greater affinity for endoglin than does TEC-4, becauseblocking of TEC-4 binding by TEC-11 was more efficient than thereciprocal event.

4. Complement fixation by TEC-4 and TEC-11 antibodies

TEC-11 fixed complement approximately 100-fold more efficiently than didTEC-4, inducing lysis of >50% of HUVEC at a concentration of 1 μg/ml(FIG. 15). TEC-4 displayed no complement fixation activity at 1 μg/mland lysed only 48% of HUVEC at a concentration of 100 μg/ml under theseconditions. The superior complement-fixing activity of TEC-11 was due,at least in part, to its greater affinity, which resulted in greaterbinding of TEC-11 than TEC-4 to HUVEC at low antibody concentrations.The concentration of TEC-11 which produced half-maximal fluorescence inFACS analyses was 0.4 μg/ml as compared with 4 μg/ml for TEC-4.

5. Reactivity of TEC-4 and TEC-11 with normal and malignant human celllines

Binding of TEC-4 and TEC-11 to endothelial cells from several speciesand a panel of non-endothelial human cell lines was determined byindirect immunofluorescence and cytofluorimetry. The results are shownin Table IV. Essentially identical results were obtained with TEC-4 andTEC-11. Substantially higher levels of staining were detected on HUVECthan on any other cell tested in this study. The specific meanfluorescence intensity (MFI) of TEC-4 and TEC-11 on HUVEC was around200.

A human endothelial cell line, ECV-304, which was derived from HUVEC(Kobayashi et al., 1991), gave lower MFI values of 35-45. ECV-304 cellsalso displayed diminished expression of several other endothelial cellmarkers, including EN4 antigen, angiotensin-converting enzyme and CD34.Endothelial cells of bovine, murine and chinese hamster origin displayedno detectable reactivity with either TEC-4 or TEC-11 antibodies. U937cells were weakly labelled. Among the tumor cells, all lymphoma/leukemia lines were negative, as were the majority (5/7) of carcinomalines. Interestingly, all 4 melanoma and sarcoma lines tested wereweakly stained by TEC-4/TEC-11, with MFI values of 12-26. In addition,2/4 breast cancer lines bound the antibodies weakly.

                  TABLE IV                                                        ______________________________________                                        Endoglin expression in tissue culture                                                                         Endoglin                                        Cell type Cell line(s) expression.sup.1                                     ______________________________________                                        Endothelial  HUVEC          ++++                                                Endothelial ECV-304 ++                                                        Endothelial BCA, CHEC, LE II, -                                                MPCE, SVEC                                                                   Myeloid U937 +                                                                Myeloid U266 -                                                                Leukemia/lymphoma ARH-77, CEM, Daudi, -                                        Gaynor L428, L540,                                                            Nalm-6, K562                                                                 Sarcoma HT-1080, SAOS-2 +                                                     Melanoma A375M, T8 +                                                          Carcinoma LOVO, NCI-H146, SCC- -                                               5                                                                            Breast tumor MDA-MB-231, SKBR3 +                                              Breast tumor MCF-7, T 47D -                                                   Fibroblast L-endoglin +++                                                     transfectant                                                                ______________________________________                                         .sup.1 Expressed as the corrected fluorescence intensity (CFI), calculate     as described in the Materials and Methods where a CFI of <5 is represente     by (-), 5-25 (+), 25-50 (++), 50-100 (+++) and >100 (++++).              

6. Correlation between endoglin expression and endothelial cellproliferation in vitro

TEC-4 and TEC-11 bound only weakly to quiescent HUVEC in situ in frozensections of human umbilical vein (see following section). Endoglinexpression reached high levels within 16 hours after the HUVEC wereremoved from the umbilical cord and placed into tissue culture and wasnot abolished in HUVEC cultures grown to confluence or deprived of serumor growth factors. However, confluent cultures displayed a bimodaldistribution of TEC-4/TEC-11 binding by FACS (FIG. 16).

To characterize HUVEC populations expressing low and high levels ofendoglin, confluent HUVEC cultures were stained with TEC-11 and sortedon a FACStar Plus cell sorter. The sorted populations were analyzed fortotal cellular protein content and relative DNA and RNA levels. Theresults of these studies are shown in FIG. 16 and Table V.

HUVEC from sparse cultures expressed uniformly high endoglin levels, asshown in FIG. 16a, hatched histogram, but when the same cells were grownto confluence and allowed to become partially quiescent over anadditional 3-6 days, a bimodal distribution of TEC-11 binding was seen(FIG. 16a, open histogram). This pattern of TEC-11 binding was notsimply a reflection of variations in cell size among the postconfluentHUVEC, because the expression of a control marker (the vitronectinreceptor) was not significantly altered between HUVEC from sparse andpostconfluent cultures (FIG. 16b). Postconfluent cells stained withTEC-11 were sorted into two fractions as indicated in FIG. 16a.Endoglin^(hi) cells had a specific MFI 4.4 times greater than that ofendoglin^(lo) cells (Table V). Similarly, there was a 1.6-fold increasein binding of free FITC to cellular proteins in permeabilizedendoglin^(hi) cells by comparison to low endoglin expressors (Table V).Upregulated protein synthesis in the endoglin^(hi) population reflectedsimilar increases in cellular transcription, as indicated by acridineorange staining. While endoglin^(lo) cells formed a single populationwith low relative RNA and DNA content (FIG. 16c), significant numbers ofendoglin^(hi) cells showed evidence of RNA and DNA synthesis consistentwith cellular activation and proliferation (FIG. 16d). Indeed, when thesorted populations were separated into zones in the dot plots (FIG.16c,d) according to standard criteria (Darzynkiewicz et al., 1976),virtually all endoglin^(lo) cells were assigned to the non-cycling (G₀)population but, by contrast, 15% and 5% of endoglin^(hi) cells werelocated in the G₁ (activated) and S+G₂ /M (proliferating) fractionsrespectively (Table V).

                  TABLE V                                                         ______________________________________                                        Colleration between endoglin expression and endothelial cell proliferation     in vitro.                                                                                Relative Stage of cell cycle (%).sup.3                            Fraction                                                                             MFI.sup.1                                                                              protein.sup.2                                                                          G.sub.0                                                                              G.sub.1 S + G.sub.2 /M                        ______________________________________                                        Endoglin.sup.lo                                                                       61.6(2.4)                                                                             100      95.8(2.4)                                                                             3.1(1.4)                                                                             1.1(1.4)                                Endoglin.sup.hi 270.2(3.8) 156 79.1(5.8  15.5(2.9) 5.4(1.2)                 ______________________________________                                         .sup.1 Mean fluorescence intensity of cells stained with 20 μg/ml          TEC11. Mean and standard deviation (in parentheses) of 2 studies.             .sup.2 Estimated from nonspecific binding of FITC to cellular proteins in     permeabilized cells.                                                          .sup.3 Estimated from 2dimensional dotplot after acridine orange staining     as described in Materials and Methods and shown in FIG. 13. Mean and          standard deviation (in parentheses) of 2 studies.                        

7. TEC-4 and TEC-11 binding to malignant and normal human tissues

a) Endothelial cells in miscellaneous tumors

TEC-4 and TEC-11 binding to vascular endothelial cells was assessed byimmunoperoxidase staining of a panel of 51 miscellaneous human tumors.The results are shown in Table VI and FIGS. 17 and 18.

Both antibodies clearly stained the cytoplasm and luminal plasmamembranes of vascular endothelial cells in a large majority of thetumors examined, including extensive series of breast and colorectalcarcinomas. TEC-4 and TEC-11 reacted with capillaries and venules butnot with arterioles in 20/22 evaluable cases. The reactivity patterns ofthe two antibodies were very similar, although TEC-11 tended to producemore intense staining than TEC-4 in some tissues (Table VI). In mosttumor samples, a large majority (80-100%) of vessels which stained withthe positive control anti-endothelial cell antibody (anti-vonWillebrands Factor) also stained moderately to strongly with TEC-4 andTEC-11. Often, TEC-4 and TEC-11 gave more uniform staining ofcapillaries than did the anti-von Willebrands antibody. TEC-4 and 11binding was variable both between and within histological tumor types.

Vascular endothelial cells were most strongly stained by TEC-4 andTEC-11 in sections of angiosarcoma, Hodgkins disease and colon, cecumand rectosigmoid carcinoma (Table VI). Parotid tumors (FIG. 17) and somebreast carcinomas (FIGS. 17 and 18) also contained heavily-labelledvessels. Moderate staining of vascular endothelium was characteristic ofpharyngeal, lung and ovarian carcinomas (Table VI). Vessels in softtissue tumors (melanoma and osteosarcoma) were stained moderately byTEC-11 but only weakly by TEC-4. Lymphoma samples examined showed littleor no staining with either antibody. TEC-4 and TEC-11 reactivity wasrestricted to human tissues--neither antibody stained endothelial cellsin a variety of mouse, rat, guinea pig and hamster tumors, nor werevessels labelled in human tumor xenografts in nude mice.

                  TABLE VI                                                        ______________________________________                                        Endoglin expression on endothelial cells in miscellaneous tumors                           Antibody                                                         Tumor type   n     anti-VWF    TEC-4 TEC-11                                   ______________________________________                                        Angiosarcoma 1     +++.sup.1   +++   +++                                        Benign breast 6 +++ +/- -                                                     tumor                                                                         Breast carcinoma 12 +++ ++ ++/+++                                             Cecum carcinoma 1 +++ +++ +++                                                 Colon carcinoma 3 +++ ++ +++                                                  Hodgkins disease 11 +++ ++ +++                                                Lymphoma 2 +++ +/- +                                                          Lung carcinoma 1 +++ ++ ++                                                    Melanoma 1 +++ + ++                                                           Osteosarcoma 1 +++ + ++                                                       Ovarian 1 +++ ++ ++                                                           carcinoma                                                                     Parotid tumor 3 +++ +++ +++                                                   Pharyngeal 2 +++ ++ ++                                                        carcinoma                                                                     Rectosigmoid 6 +++ ++/+++ +++                                                 carcinoma                                                                   ______________________________________                                         .sup.1 Staining intensity was strong (+++), moderate (++), weak (+) or        negative (-).                                                            

b) Endothelial cells in normal tissues

A panel of 27 normal human tissues was used to assess the reactivity ofTEC-4 and TEC-11 with vascular endothelial cells in non-malignantsettings. The staining conditions used were ones that gave distinctstaining of tumor vasculature. As shown in Table VII, the staining ofnormal endothelium obtained with both antibodies was usually weak ornegative, but there was moderate staining of capillary endothelium inadrenal gland and placenta (both antibodies), parathyroid (TEC-4) andlung, cervix, testis, kidney and lymphoid organs (TEC-11). In addition,TEC-4 displayed strong binding to skin vessels (Table VII). Endothelialcells in numerous tissues showed no detectable staining with one or bothantibodies, including bladder, brain, cranial nerve, mammary gland (FIG.18b), ovary, pancreas, stomach, thymus and umbilical vein (FIG. 17d).

Several `normal` tissue samples were in fact adjacent non-malignanttissue from cancer biopsies, enabling staining of endothelial cells innormal and malignant areas of the same organ to be compared in the samesection. FIG. 17 (a and b) shows a sample of parotid tumor andassociated histologically normal parotid gland where a marked differencein the staining of vascular endothelial cells by TEC-4 antibody isvisible. In the stroma between the nests of malignant cells, allendothelial cells were heavily labelled (FIG. 17a). By contrast, onlylight staining of a single vessel in the normal glandular tissue wasdiscernible (FIG. 17b).

                  TABLE VII                                                       ______________________________________                                        Endoglin expression on endothelial                                              cells in non-neoplastic tissues                                                           Antibody                                                                          anti-vWF    TEC4   TEC11                                    ______________________________________                                        Normal tissues                                                                  Adrenal +++.sup.1 ++ ++                                                       Bladder ++ - -                                                                Brain cortex ++ - -                                                           Brain stem ++ - -                                                             Cerebellum +++ - -                                                            Colon +++ ++ +                                                                Cranial nerve +++ - -                                                         Gall bladder +++ ± +                                                       Kidney +++ ± ±                                                          Liver +++ + +                                                                 Lung +++ ± ++                                                              Mammary gland +++ - -                                                         Ovary +++ ± -                                                              Pancreas + - -                                                                Parathyroid ++++ ++ -                                                         Parotid gland +++ ± ±                                                   Placenta +++ ++ ++                                                            Prostate +++ ± ±                                                        Salivary gland +++ ± ±                                                  Skin +++ +++ +                                                                Stomach +++ +/- +                                                             Stomach muscle +++ - +                                                        Testis +++ - ±                                                             Thymus +++ - -                                                                Thyroid +++ + ±                                                            Tonsil +++ + +                                                                Umbilical vein +++ +/- +/-                                                    Inflammatory tissues                                                          Tonsilitis +++ ++ +++                                                         Reactive +++ +/- +                                                            hyperplasia                                                                   Cat-scratch +++ + ++                                                          fever                                                                         colitis +++ ++ +++                                                          ______________________________________                                         .sup.1 Staining intensity was strong (+++), moderate (++), weak (+) or        negative (-).                                                            

c) Endothelial cells during tumor progression in the breast

Endothelial cells in normal mammary glands were not stained by eitherTEC-4 or TEC-11 (FIG. 18b) whereas endothelial cells in malignant breasttumors were stained moderately to strongly by both antibodies (FIG. 17cand Table VI). A series of abnormal breast tissues was thereforeexamined to determine at which stage of neoplastic progression endoglinexpression was initiated. Vessels in 6/6 benign fibroadenomas displayedlittle or no reactivity with TEC-4 and TEC-11 (Table VI), as did thosein low-grade hyperplastic lesions and early carcinoma-in-situ. Moderatestaining was apparent in late-stage intraductal carcinomas whereas allvessels in frank malignant carcinomas were heavily labelled with bothTEC-4 and TEC-11 (FIG. 17c, FIG. 18d, Table VI).

d) Endothelial cells in inflammatory sites

TEC-4 and TEC-11 staining of endothelial cells was strong in 3/4 typesof inflammatory tissues examined (Table VII). TEC-11 staining wassomewhat stronger than TEC-4 staining in all cases. In 6/8 cases oftonsillitis, cat-scratch fever and ulcerative colitis, which are allassociated with neovascularization, endothelial cells were stainedmoderately to strongly with TEC-11. By contrast, vessels were weaklystained in 2 cases of reactive hyperplasia, a non-angiogenic condition.

e) Non-endothelial cells

Both TEC-4 and TEC-11 showed highly restricted binding to cryostatsections of normal and malignant human tissues, reacting primarily withvascular endothelial cells. However, both antibodies displayedcross-reactivities, typically quite weak, with certain non-endothelialcell types (Table VIII). Both antibodies bound weakly to stromalcomponents in the prostate, the basal layer of seminiferous tubules, andto follicular dendritic cells in lymphoid organs and in small lymphoiddeposits associated with colorectal tumors (Table VIII). Strong stainingof syncytiotrophoblast in placenta was also seen. TEC-11 gave a morerestricted staining pattern than did TEC-4 (Table VIII), which alsobound to myoepithelial cells in the breast, smooth muscle cells,especially in the gut, and to miscellaneous epithelial tissues includingsome rectal glandular epithelium, epidermis and breast carcinoma cellsin a minority of samples.

7. Selective cytotoxic effects of TEC-11 immunotoxin

An immunotoxin was prepared by chemical linkage of TEC-11 antibody withdeglycosylated ricin A-chain, as described in detail in Example II. Theimmunotoxin was tested for cytotoxicity against quiescent, confluent andsubconfluent populations of human endothelial cells in a standardprotein synthesis inhibition assay, as described in Example II. Forcomparison, the same endothelial cell cultures were also treated with anisotype-matched non-binding immunotoxin (MTSA-dgA), an immunotoxinagainst an endothelial cell antigen (ICAM-1) whose expression does notvary according to the growth status of the cells (UV-3-dgA) and nativericin.

The results of several such assays are combined and shown in FIG. 19 andTable IX. The negative and positive controls, MTSA-dgA (FIG. 19a) andricin (FIG. 19c), respectively, gave essentially identical cytoxicityprofiles against all three endothelial cell populations. Ricin inhibitedprotein synthesis by 50% (IC₅₀) in quiescent, confluent and subconfluentcultures at concentrations of 0.15-0.27 pM (FIG. 19c; Table IX) andMTSA-dgA displayed no cytotoxicity to any endothelial cell population atconcentrations below 0.1 μm (FIG. 19a and Table IX).

The anti-ICAM-1 immunotoxin, UV-3-dgA, was weakly but clearly cytotoxicto endothelial cells at all stages of growth, with IC₅₀ values rangingfrom 9 to 80 nM (FIG. 19b and Table IX). As expected from their highermetabolic rate and consequent requirements for protein synthesis, theproliferating subconfluent cultures were almost 9-fold more sensitive toUV-3dgA than were the quiescent cells (Table IX). The confluent cultureswere fully metabolically active but were 4-fold less sensitive toUV-3-dgA than were the subconfluent cells (Table IX), probablyreflecting decreased accessibility of the target antigen due to thedense packing of the cells in confluent cultures.

By contrast, TEC-11-dgA showed striking differences in cytotoxicitytowards quiescent, confluent and subconfluent endothelial cells (FIG.19d and Table IX). TEC-11-dgA at a concentration of 10-8M inhibitedprotein synthesis in quiescent cultures by only 10l and in confluentcultures by only 20%, (FIG. 19d) and yet inhibited protein synthesis insubconfluent cultures by over 60% at concentrations as low as 10⁻¹⁰ M(FIG. 19d). When the IC₅₀ values for TEC-11-dgA towards the differentendothelial cell cultures were compared, it was found that theimmunotoxin was 2400-fold more toxic to subconfluent cells than toconfluent cultures. When sparse and quiescent cells were compared, thisratio rose to over 3000 (Table IX).

Taken together, these results demonstrate that an immunotoxin preparedfrom the TEC-11 antibody displays highly selectively cytotoxicitytowards subconfluent, actively proliferating human endothelial cells.

                  TABLE VIII                                                      ______________________________________                                        Reactivity of TEC-4 and TEC-11 with non-endothelial cells                                                 Antibody                                                     Cell type   TEC-4     TEC-11                                       ______________________________________                                        Tumor tissue                                                                    Angiosarcoma Sarcoma ++  ++                                                   Breast carcinoma Carcinoma + -                                                 Myoepithelium ++ -                                                           Colorectal carcinoma Smooth muscle +/++ -                                     Hodgkins disease Follicular dendritic + +                                      cells                                                                        Various Stroma +/- +/-                                                        Normal tissue                                                                 Gut Smooth muscle +/++ -                                                      Liver Bile duct + -                                                           Lymphoid tissues Follicular dendritic + +                                      cells                                                                        Mammary gland Myoepithelium ++ -                                              Placenta Syncyhotrophoblast ++ ++                                             Prostate Fibromuscular stroma ++ +                                            Skin Epidermis +/- -                                                          Testis Basal layer of + +                                                      seminiferous tubules                                                       ______________________________________                                         .sup.1 Staining intensity was strong (+++), moderate (++), weak (+) or        negative (-).                                                            

                  TABLE IX                                                        ______________________________________                                        Immunotoxin cytotoxicity to endothelial cells at different stages of          growth.                                                                               IC.sub.50 (nm)                                                        Quiescent    Confluent Subconfluent                                                                            IC.sub.50 ratio                              (Q)          (C)       (S)       Q/S   C/S                                    ______________________________________                                        Treatment                                                                       MTSA-dgA 80 1601 45 1.8 3.6                                                   Ricin 0.00027 0.00018 0.00015 1.8 1.2                                         UV-3-dgA 37 80 9 4.1 8.9                                                      TEC-11-dgA 230.sup.2 180.sup.2 0.075 3067 2400                              ______________________________________                                         .sup.1 Extrapolated by fitting an exponential curve to the graph shown in     FIG. 19a.                                                                     .sup.2 Extrapolated by fitting an exponential curve to the graph shown in     FIG. 19d.                                                                

C. Discussion

In this example, the inventors describe two new monoclonal antibodies,TEC-4 and TEC-11, directed against a marker that is upregulated intumor-associated vascular endothelial cells. TEC-4 and TEC-11specifically react with endoglin, which is known to be aproliferation-linked endothelial cell marker that is upregulated ondividing endothelial cells in vitro and on vascular endothelial cells insolid tumors, sites of chronic inflammation and fetal placenta in vivo.

HUVEC rapidly express endoglin after removal from the umbilical cord andestablishment in tissue culture. Induction of the antigen was notdiminished by depriving the cells of serum or growth factors orincreased by addition of cytokines, such as IL-1, TNF-α and IFN-γ, knownto activate endothelial cells (Dustin et al., 1986; Rice et al., 1990),in accordance with the findings of Westphal et al. (Westphal et al.,1993). The level of endoglin expression appeared to correlate with entryinto or progression through the cell cycle. In HUVEC which had beengrown to confluence, two subpopulations were present, one with lowendoglin levels and the other with high expression. All endoglin^(lo)cells were in G₀, but the endoglin^(hi) population contained significantproportions of activated (G₁) and dividing (S+G₂ M) cells, as indicatedby increased levels of protein/RNA and DNA, respectively. The majorityof endoglin^(hi) cells were also in G₀, suggesting that cell surfaceendoglin is long-lived and is maintained at high levels in cells thathave divided and subsequently enter a non-cycling state.

An association between increased endoglin expression and endothelialcell proliferation is also suggested by strong TEC-4/TEC-11 staining ofblood vessels in sites of neovascularization. Moderate to stronglabelling of most or all capillaries and venules was observed incryostat sections of solid tumors of diverse histological types. Theonly malignant tumors that did not show significant endothelial cellstaining were B-lymphomas, in keeping with the fact that lymphomas,unlike carcinomas and non-lymphoid sarcomas, grow by infiltratingexisting vascular tracts rather than by inducing de novo blood vesselgrowth (Denekamp and Hobson, 1982). Endothelial cell staining was absentor weak in all normal healthy tissues examined other than placenta,where endothelial cells proliferate even faster than they do in tumors(Denekamp, 1986; Denekamp and Hobson, 1982). The inventors also observedendoglin upregulation in several cases of ulcerative colitis, a chronicinflammatory condition, and in cat-scratch fever and tonsillitis, whichare associated with marked vascular proliferation (Garcia et al., 1990;Fujihara, 1991). By contrast, endoglin levels in reactive hyperplasia,which is not associated with angiogenesis (Jones et al., 1984), were nothigher than in normal lymph nodes. Increased endoglin expression byvascular endothelial cells has also been reported inangiogenesis-dependent chronic inflammatory skin lesions, such aspsoriasis, dermatitis and granulation tissue, and on one case ofcutaneous malignant melanoma (Westphal et al., 1993). Increased endoglinexpression appeared to be related to endothelial proliferation ratherthan to inflammation per se, because a range of inflammatory cytokineshad little or no effect on endoglin levels in HUVEC in vitro (Westphalet al., 1993).

The reactivity patterns of TEC-4 and TEC-11 in frozen sections of humantissues and on human cells in vitro were similar to those of otheranti-endoglin antibodies (Gougos and Letarte, 1988; Gougos et al., 1992;O'Connel et al., 1992; Buhring et al., 1991; Westphal et al., 1993).TEC-4 and TEC-l bound to HUVEC and U937 cells in vitro, but wereunreactive with the human lymphoma lines K562, CEM and Daudi, inaccordance with previous reports (Gougos and Letarte, 1988; Westphal etal., 1993). All the antibodies labelled endothelial cells inmiscellaneous human tissues and gave strong staining of fetalendothelium and syncytiotrophoblast in the placenta (Gougos et al.,1992; Westphal et al., 1993).

Several authors have reported stronger staining of endothelial cells innormal organs, especially kidney and liver (Gougos and Letarte, 1988;Westphal et al., 1993) and umbilical cord (Gougos and Letarte, 1988),than the inventors observed with TEC-4 and TEC-11. These discrepanciesprobably reflect differences in the sensitivity of theimmunohistochemical staining techniques employed in differentlaboratories. In the present study, staining conditions were selectedwhich produced clear staining of tumor endothelium whereas in previousstudies (Gougos and Letarte, 1988; Gougos et al., 1992), more sensitivestaining conditions were previously used in order to visualize normalendothelium. Evidence that this is indeed true is provided by ourfinding that 44G4, previously reported to stain normal endothelium,produced identical endothelial staining patterns to that describedherein for TEC-4 and TEC-11 when the current techniques were employed.However, TEC-4 and TEC-11 recognize a distinct epitopes from that of44G4, as shown by the failure of 44G4 to block TEC-4 or TEC-11, even athigh ratios.

TEC-11 showed almost complete specificity for endothelial cells whereasTEC-4 also reacted moderately strongly with certain non-endothelialcells, particularly smooth muscle. The inventors interpret theseadditional reactivities of TEC-4 as being cross-reactivities possiblyrelated to the low affinity of TEC-4. Similar staining of smooth musclewas frequently seen during the screening of the original hybridomasupernatants on tissue sections.

Upregulation of endoglin on vascular endothelial cells in solid tumorsand chronic inflammatory disorders might be involved functionally in theregulation of angiogenesis in these pathological conditions. Recentevidence indicates that endoglin is an essential component of the TGF-β(transforming growth factor-β) receptor complex of human endothelialcells. It binds TGF-β1 and TGF-β3 with a K_(D) =50pM (Cheifetz et al.,1992). TGF-β inhibits endothelial cell proliferation in vitro (Madri etal., 1992) but, paradoxically, is a potent angiogenic agent in vivo(Enenstein et al., 1992). Increased expression of endoglin byproliferating endothelial cells could modulate their response to TGF-βand hence regulate the angiogenic process (Cheifetz et al., 1992).

Antibodies to antigens other than endoglin have been reported to stainendothelial cells in miscellaneous neoplasms but not those in normaltissues. EN7/44 reacts with a predominantly intracellular antigen (Mr30.5 kDa) in budding capillary sprouts in solid tumors and otherneovascular sites whose expression appears to be linked to migrationrather than proliferation (Hagemeier et al., 1986). FB-5 recognizes aheavily sialylated glycoprotein (Mr 170 kDa) on reactive fibroblasts andin a proportion of blood vessels in various human tumors (Rettig et al.,1992). E9 reacts with a 95 kD homodimer that is upregulated in tumors,fetal organs and regenerating tissues, but which can be distinguishedfrom endoglin on the basis of lack of reactivity with placentalendothelium and different staining of tumor-derived endothelial cells invitro (Wang et al., 1993). Taken overall, the uniformity of staining ofvessels in different tumors and within any individual tumor suggest thatTEC-4 and TEC-11 compare favorably with these antibodies.

The TEC-4 and TEC-11 antibodies have important potential for diagnosisand therapy of human cancer. Firstly, the antibodies could be used todistinguish between histologically indistinct benign and malignantlesions. Studies in breast (Weidner et al., 1992; Horak et al., 1992),prostate (Bigler et al., 1993), bladder (Bigler et al., 1993), andcervical (Sillman et al., 1981) carcinomas have established that highvessel density or tumor angiogenic activity is strongly correlated withrisk of metastasis and poor prognosis and so could be used to determinewhen aggressive post-operative therapy is appropriate (Weidner et al.,1992; Horak et al., 1992). Diagnosis in these studies required laboriousenumeration of capillaries labelled with pan- endothelial cell markers(Weidner et al., 1992; Horak et al., 1992; Bigler et al., 1993) or theuse of complex and subjective in vivo assays of angiogenesis (Chodak etal., 1980), both of which might be supplanted by a simpleimmunohistochemical procedure employing TEC-4 or TEC-11. Indeed, thestudies of breast tumors reported in this Example indicate that vascularendothelial cell expression of endoglin, as determined using TEC-4 orTEC-11, may distinguish between intraductal carcinoma in situ (CIS), anaggressive preneoplastic lesion and lobular CIS, which is associatedwith a more indolent clinical course.

Secondly, the antibodies could be used for imaging tumors in cancerpatients. Being present on the luminal face of the endothelial cell,endoglin is ideally situated for antibody binding and should thereforepermit rapid imaging. The antibodies would not be subject to the majorlimitation of imaging procedures against the tumor cells themselves,since they need not penetrate into solid tumor masses. In addition,being of the IgM isotype, extravasation of TEC-4 and TEC-11 should beminimal and their specific imaging of antigens in the intravascularcompartment should be superior.

Thirdly, the antibodies could be used for therapy. The highly accessiblelocation of endoglin on the luminal surface of the tumor vasculature isespecially advantageous for therapeutic application, because all of thetarget endothelial cells are able to bind the therapeutic antibody, asshown in Example I. Both TEC-4 and TEC-11 are complement-fixing and somight induce selective lysis of endothelial cells in the tumor vascularbed. Also, the antibodies could be used to deliver therapeuticquantities of radioisotopes, toxins, chemotherapeutic drugs orcoagulants to the tumor vasculature. Animal studies indicates thatanti-tumor endothelial cell immunotoxins are most effective whencombined with anti-tumor cell immunotoxins, which kill those tumor cellsthat have invaded surrounding normal host tissue (as disclosedhereinabove and in Burrows and Thorpe, 1993). Thus, TEC-4 or TEC-11could be used clinically in combination with antibodies againstwell-characterized tumor markers such as p185^(HER-2), TAG-72, andCO17-1A (Shepard et al., 1991; Greiner et al., 1991; Kaplan, 1989) orindeed with conventional chemotherapeutic drugs.

EXAMPLE V Preparation, Characterization and Use of Antibodies DirectedAgainst Tumor-Derived Endothelial Cell Binding Factors

This example describes the generation of polyclonal and monoclonalantibodies directed against tumor-derived endothelial cell "bindingfactors" for use in distinguishing between tumor vasculature and thevasculature of normal tissues. Particularly described is the generationof antibodies directed against vascular permeability factor (VPF), alsotermed vascular endothelial cell growth factor (VEGF), and against bFGF(basic fibroblast growth factor).

For further details concerning FGF one may refer to Gomez-Pinilla andCotman (1992); Nishikawa et al. (1992), that describe the localizationof basic fibroblast growth factor; Xu et al. (1992), that relates to theexpression and immunochemical analysis of FGF; Reilly et al. (1989),that concerns monoclonal antibodies; Dixon et al. (1989), that relatesto FGF detection and characterization; Matsuzaki et al. (1989), thatconcerns monoclonal antibodies against heparin-binding growth factor;and Herbin and Gross (1992), that discuss the binding sites for bFGF onsolid tumors associated with the vasculature.

In these studies, rabbits were hyperimmunized with N-terminal peptidesof human VEGF, mouse VEGF, guinea pig VEGF, human bFGF, mouse bFGF orguinea pig bFGF coupled to tuberculin (purified protein derivative, PPD)or thyroglobulin carriers. The peptides were 25 to 26 amino acids inlength and were synthesized on a peptide synthesizer with cysteine asthe C-terminal residue. Antisera were affinity purified on columns ofthe peptides coupled to Sephraose matrices.

Antibodies to VEGF were identified by ELISA and by their stainingpatterns on frozen sections of guinea pig tumors and normal tissues.Polyclonal antibodies to guinea pig VEGF and human VEGF reacted with themajority of vascular endothelial cells on frozen sections of guinea pigL10 tumors and a variety of human tumors (parotid, ovarian, mammarycarcinomas) respectively. The anti-human VEGF antibody stained mesangialcells surrounding the endothelial cells in normal human kidneyglomerulae and endothelial cells in the liver, but did not stain bloodvessels in normal human stomach, leg muscle and spleen. The anti-guineapig VEGF antibody did not stain endothelial cells in any normal tissues,including kidney, brain, spleen, heart, seminal vesicle, lung, largeintestine, thymus, prostrate, liver, testicle and skeletal muscle.

Polyclonal antibodies to human FGF stained endothelial cells in parotidand ovarian carcinomas, but not those in mammary carcinomas. Anti-humanFGF antibodies stained glomerular endothelial cells in human kidney, butnot endothelial cells in normal stomach, leg muscle and spleen.

Monoclonal antibodies to guinea pig VEGF, human VEGF and guinea pig bFGFwere prepared by immunizing BALB/c mice with the N-terminal sequencepeptides (with cysteine at the C-terminus of the peptide) coupled to PPDor to thyroglobulin. The synthetic peptides immunogens of definedsequence are shown below and are represented by SEQ ID NO:l, SEQ ID NO:2AND SEQ ID NO:3, respectively:

    ______________________________________                                        guinea                                                                              A P M A E G E Q K P R E V V K F M D V Y K R S Y C                         pig                                                                           VEGF                                                                          human A P M A E G G G Q N H H E V V K F M D V Y Q R S Y C                     VEGF                                                                          guinea M A A G S I T T L P A L P E G G D G G A F A P G C                      pig                                                                           bFGF                                                                        ______________________________________                                    

The peptides were conjugated to thyroglobulin or to PPD by derivatizingthe thyroglobulin with succimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and reacting thederivative with the peptide. This yields a conjugate having one or morepeptide sequences linked via a thioether bond to thyroglobulin.

Specifically, the generation of monoclonal antibodies against the abovesequences was achieved using the following procedure: BALB/c mice wereimmunized by serial injections with peptide-PPD or peptide-thyroglobulininto several sites. Four or five days after the last injection, thespleens were removed and splenocytes were fused with P3×G3Ag8.653myeloma cells using poyelthyleneglycol according to the procedurespublished in Morrow, et al. (1991). Individual hybridoma supernatantswere screened as follows:

First screen: ELISA on peptide-thyroglobulin-coated plates.

Second screen: ELISA on cysteine linked via SMCC to thyroglobulin.

Third screen: Indirect immunoperoxidase staining of frozen sections ofguinea pig line 10 tumor or human parotid carcinoma.

Fourth screen: Indirect immunoperoxidase staining of frozen sections ofmiscellaneous malignant and normal guinea pig and human tissues.

Antibodies were selected that bound to peptide-thyroglobulin but not tocysteine-thyroglobulin, and which bound to endothelial cells inmalignant tumors more strongly than they did to endothelial cells innormal tissues (Table X).

                  TABLE X                                                         ______________________________________                                        Reactivity of Monoclonal Antibodies                                                           Reactivity with Tumor                                                                       Tumor                                                         Endothelium Reactivity                                          MoAB  Immunogen.sup.+                                                                         Class   g. pig human  Pattern*                                ______________________________________                                        GV14  gp VEGF   IgM     +      +      BV + some                                      tumor cells                                                              GV35 gp VEGF IgM ± ± Tumor cells,                                            weak on BV                                                               GV39 gp VEGF IgM + + BV and some                                                   tumor cells                                                              GV59 gp VEGF IgM + + BV and some                                                   tumor cells                                                              GV97 gp VEGF IgM + + BV, weak on                                                   tumor cells                                                              HV55 hu VEGF IgG ? + Basement                                                      membrane,                                                                     some BV                                                                  GF67 gp FGF IgM + + BV and tumor                                                   cells                                                                    GF82 gp FGF IgM + + BV and tumor                                                   cells                                                                  ______________________________________                                         *BV = blood vessels                                                           .sup.+ gp = guinea pig                                                        hu = human                                                                    GV97 Staining of Human and Guinea Pig Tissue Sections                    

The GV39 GV97 antibodies against guinea pig VEGF N-terminus bound toendothelial cells in miscellaneous human malignant (Table XI) and normal(Table XII) tissues. The GV39 and GV97 antibodies were deposited Dec.12, 1997 with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, USA and given the ATCC Accession numbersATCC HB-12450 and ATCC HB-12451, respectively.

Binding to endothelial cells in malignant tumors tended to exceed thatto endothelial cells in normal tissues; however, this difference was notstriking.

The staining of endothelial cells in guinea pig tumor (line 10hepatocellular carcinoma) and normal tissues was similar in distributionand intensity to that observed with human tissues (Table XIII).

In Tables XI through XV, + indicates a positive, as opposed to anegative, result. The numbers 2+, 3+and 4+ refer to a positive signal ofincreasing strength, as is routinely understood in this field of study.

                                      TABLE XI                                    __________________________________________________________________________    Anti-GPVEGF on Human Tumors                                                                 20 Purified GV97                                                                          1 μg/ml or                                                                        0.5 GV97         GV59                        Tumor TISSUE  μg/ml                                                                         10 μg/ml                                                                        5 μg/ml                                                                        2 μg/ml                                                                           μg/ml                                                                          supt.                                                                              GV14                                                                              GV39                                                                              supt.                       __________________________________________________________________________    DIGESTIVE TRACT                                                                 92-01-A073  2+  1+ +/-                                                       ve   4+ 4+                                                                     esophagus carcinoma                                                           M4 Parotid      4+                                                            87-07-A134 Parotid  3+ 2+ +/-                                                ve   3+ 4+                                                                     carcinoma                                                                     M5 Parotid      4+                                                            88-04-A010 parotid  1-2+ 1+                                                  ve                                                                            ve    1-3+                                                                     adenoca.                                                                      90-11-B319 Adeno. Ca.      3-4+  3-4+                                         of colon to liver                                                             94-02-B021C      3-4+  3-4+                                                   Adenocarcinoma of                                                             colon                                                                         93-10-A333 Adeno. Ca. 4+ 2-4+ 1-4+                                           ve-1+  4+   3+                                                                 of colon with normal                                                          93-02-B004 Villous and 4+ 3-4+ 2-4+ 1-2+  3-4+  2-3+                          Adenomatous polyp of                                                          colon                                                                         93-02-A130  3+ 2+ +/--1+                                                     ve 4+  4+ 3-4+                                                                 Leiomyosarcoma in                                                             colon                                                                         93-02-B020 Gastric Ca.  4+ 2+ 2-3+                                           ve-1+   1-2+                                                                   93-04-A221 Pancreas  3-4+ 2-3+ 1-2+                                          ve-   4+ 4+                                                                    Adenoca.     0.5+                                                             94-04-A390 rectum  4+ 3+ 1-2+ 1+    3+                                        adenoca.                                                                      93-12-A160 tongue  1-2+ +/-                                                  ve                                                                            ve   3+ 3+                                                                     carcinomaadenoca.                                                             101-84a Stomach signet  3+ 2+                                                ve-1+                                                                         ve most 1-   3+                                                                ring Ca. (101-84b      2+ but a                                               pair)      few 3-4+                                                           90-05-A172 Stomach  4+ 3+ 1-2+                                               ve-1+                                                                         ve 3+                                                                          Adenoca.                                                                      REPRODUCTIVE TRACT                                                            91-10-A115 Squam. cell 1-4+ 1-3+ 1-2+ 1-2+  1-4+   1-3+                       Ca. of vulya                                                                  93-03-A343 Prostate  +/- - +/- +/- to 1-2+ +/-   3-4+ 3-4+                    Adenoca.  3-4+ to 2-3+                                                        MUSCLE                                                                        IMMUNE SYSTEM                                                                 URINARY SYSTEM                                                                93-10-B002 Renal cell      2+   3+                                            Ca.                                                                           90-01-A225 Renal cell  4+ 4+ 3-4+ of most 1-3+ of 3-4+  3+ 3-4+                                                                Ca.     some                 93-01-A257 Transit. 3-4+ 2-3+ 1-2+ +/-  2-3+   2-3+                           cell Ca. of bladder                                                           ENDOCRINE SYSTEM                                                              94-01-A246 4+ 4+ 3-4+ 3+  4+  3-4+                                            Pheochromocytome of                                                           adrenal                                                                       93-11-A074 Adrenal 3-4+ 3-4+ 2-3+ 1+  3-4+   4+                               Cort. Ca.                                                                     RESPIRATORY SYSTEM                                                            93-08-N009 Lung      3-4+  3-4+ 3-4+                                          Adenoca.                                                                      92-10-A316 Sq. cell  4+ 3-4+ 1-2+                                            ve-   4+ 4+                                                                    lung Ca.     0.5+                                                             03-05-A065 Lung  4+ 3-4+                                                     ve-1+ 1+   3+ 3+                                                               adenoca.                                                                      CENTRAL NERVOUS                                                               SYSTEM                                                                        94-01-A299 malig. 4+ 4+ 4+ 3-4+  4+  3-4+                                     metast. schwanoma to                                                          Lymph node                                                                    92-10-A139 Meningioma 4+ 3-4+ 2-3+ 1-2+  4+   3-4+                            91-12-A013 Meningioma 4+ 2-3°                                         ve-3+ +/-  4+   3+                                                             93-03-A361 Atypical  4+ 4+ 3+ 2+ 4+   3+                                      meningioma                                                                    INTEGUMENTARY SYSTEM                                                          94-04-V037 Skin Sq.                                                          ve to                                                                         ve to                                                                         ve to 1+                                                                      ve   2-3+ 2-3+                                                                 cell Ca. w/normal   3+                                                        89-02-225 leg sarcoma  4+ 3-4+ 1+ 1+   4+ 2+                                  MISC. TUMORS                                                                __________________________________________________________________________

                                      TABLE XII                                   __________________________________________________________________________    Anti-GPVEGF On Human Normal Tissues                                                                            1 μg/ml                                                 20   Purified GV97 or   0.5   GV97           GV59               Tumor TISSUE  μg/ml                                                                           10 μg/ml                                                                          5 μg/ml                                                                           2 μg/ml                                                                         μg/ml                                                                            supt. GV14                                                                              GV39 supt.              __________________________________________________________________________    DIGESTIVE TRACT                                                                 91-01-A128  3+  2+ 1+                                                        ve   2-3+ 2-3+                                                                 Bladder w/                                                                    cystitis                                                                      94-02-B020      2-3+  2-3+                                                    uninvolved colon                                                              92-01-A292 N. 4+ 4+ 4+ 3-4+  4+   3-4+                                        Colon                                                                         93-10-A116 N. Z-4+ 1-4+ 1-3+                                                 ve-2+                                                                         ve 3-4+  2-3+ 3-4+                                                             Colon                                                                         90-06-A116 N.      3+ of many  2+                                             colon                                                                         93-02-A350 N.  3-4+ 3+ 1+ +/-   4+ 4+                                         esophagus                                                                     93-05-A503 N.      4+   4+                                                    Ileum                                                                         94-03-A244 N.  4+ 1-3+                                                       ve-1+                                                                         ve   4+ 4+                                                                     Liver                                                                         90-02-B132 N. 1+ of a +/-                                                    ve                                                                            ve                                                                            ve 1-3+ 2-3+ 2-3+ 2-3+                                                         Liver few                                                                     94-01-A181 N.  1-4+ 1-3+ 1-3+ of a                                           ve    3-4+                                                                     Pancreas    few                                                               90-05-D008 N.  2-4+ 1-3+ +/-                                                 ve   2-3+ 2-3+                                                                 Pancreas                                                                      93-05-A174 N.  2+ of a few 1-2+ of a 1+ of a                                 ve                                                                            ve  3+ of a 2-3+                                                               Parotid   few few    few                                                      94-04-A391 N.  1-3+                                                          ve-2+                                                                         ve                                                                            ve    3+                                                                       Small bowel                                                                   88-06-107 N. 3+ 2+ +/-                                                       ve  3-4+   3+                                                                  Stomach                                                                       101-84b N.  3-4+ in main 2-3+ in +/- in                                      ve in main 3+   3-4+                                                           Stomach (101 = 84a  and main and main and and 1+ in                           pair)  periphery 3-4+ in 2+ in periphery                                         periphery periphery                                                        90-11-B337 N.  2-3+ +/--1+                                                   ve                                                                            ve   3+ 3+                                                                     Stomach                                                                       REPRODUCTIVE TRACT                                                            93-04-A041 N.      4+  3+                                                     Breast                                                                        94-02-A197 N.      4+  3+                                                     Breast                                                                        w/fibrocystic                                                                 change                                                                        93-02-A051 Breast                                                            ve-1+                                                                         ve                                                                            ve                                                                            ve   +/- +/- -2+                                                               w/fibrocystic                                                                 change                                                                        93-02-A103 Breast 4+ 3+ 2+ 1+                                                 w/fibrocyst                                                                   change                                                                        92-11-A006 N. 2+ of 1-2+ of most 0.5+                                        ve                                                                            ve 1-2+ of   3+ of                                                             ectocervix most     some   most                                               91-03-/A207 N.  2.5+ 1.5+ 1+ .5+    2-3+                                      ectocervix                                                                    92-02-A139 N. 1+ in most                                                     ve in most                                                                    ve                                                                            ve                                                                            ve in                                                                         ve in                                                                          ovary w/corp. but 2+ in but 1+ in    most but   most                          lusteum one area one area    3-4+ in   bet 3-4                                      one area   in one                                                                area                                                                 93-0G-A11B N.  1+ of a few                                                   ve                                                                            ve                                                                            ve    3+                                                                       Prostate                                                                      93-11-A317d  3-4+ 2-3+                                                       ve-3+                                                                         ve-1+   3-4+ 3-4+                                                              Prostate chip                                                                 93-02-A315  0.5-1+ 0.5+                                                      ve                                                                            ve   1+ 1.2+                                                                   Seminal Vesicle                                                               92-04-A069 N.  1+ +/- +/- +/-   1-2+                                          testis                                                                        91-04-A117 Ureter  1+ +/-                                                    ve                                                                            ve   +/--1+ 3-4+                                                               w/inflammation                                                                MUSCLE                                                                        94-01-A065 N.  3-4+ 2+ +/-                                                   ve   3-4+ 4+                                                                   Heart                                                                         91-07-D007 N. 1-4+ 1-3+ 1-2+                                                 ve                                                                            ve 1-3+   1-3+                                                                 skeletal muscle                                                               95-03-A395 N.  4+ 3-4+ 1-2+ 0.5-1+   4+ 4+                                    Skeletal muscle                                                               IMMUNE SYSTEM                                                                 90-01-A077 N. 2-3+ 2+ 1+ of some                                             ve                                                                            ve 2-3+   3-4+                                                                 lymph node                                                                    90-08-A022 N.  most 1+ but most 0.5+ most -ve most -ve   3+ 3+                lymph node  a few 4+ but a few but a few but a few                               2+ 2+ 0.5-1+                                                               91-03-A057 N.  2+ 1+ +/-                                                     ve   3-4+ 3-4+                                                                 lymph node                                                                    91-09-B017E  3+ 2+ +/--1+                                                    ve   2-3+ 2-3+                                                                 uninvolved lymph                                                              node                                                                          93-07-A236 N.  3-4+ 3-4+                                                     ve-3+                                                                         ve    2-4+                                                                     specin                                                                        93-07-252 N.  3+ 1+ +/-                                                      ve   2-3+                                                                      specin                                                                        ENDOCRINE SYSTEM                                                              94-04-A252 N. 4+ 4+ 3-4+ 1-2+  4+   3+                                        adrenal w/                                                                    medulia and                                                                   cortex                                                                        93-05-A086 N.  most -ve a most -ve a                                         ve                                                                            ve   2-3+ 3-4+                                                                 Adrenal medulla  few 1-2+ few 1-2+                                            92-03-A157  1+ +/- +/-                                                       ve   4+ 4+                                                                     Hyperplasic                                                                   thyroid                                                                       91-03-B019 N.                                                                ve-3+                                                                         ve-2+                                                                         ve-1+                                                                         ve   2-3+ 2-3+                                                                 Thyroid                                                                       URINARY SYSTEM                                                                93-09-A048 N.      4+   2-3+                                                  Kidney                                                                        91-11-A075 N.  4+ 3+ 2+ 1+ 4+ on  4+ on 4+ on                                 Kidney      glomeruli  glomerul glomeru                                               i li                                                                  93-10-B001 N.  4+ 3+ +/-                                                     ve 4+ on  4+ on 4+ on                                                          Kidney      glomeruli  glomerul glomeru                                               i li                                                                  INTEGUMENTARY SYSTEM                                                          92-08-A029 N.  +/- to 4+ +/- to 3+ +/- to 1+ +/-   2+ 2+                      Breast skin                                                                   89-02-257  4+ 3-4+ 2-3+ 1-2+   1+ 3-4+                                        cartilage                                                                     marches 2SS                                                                   RESPIRATORY SYSTEM                                                            93-05-A203 N.                                                                ve-2+                                                                         ve-1+ +/-                                                                     ve  2+ 3+                                                                      Lung                                                                          92-12-A263 N.  2-3+ w/ducts 1-2+ w/                                          ve                                                                            ve    2-3+                                                                     Bronchus  staining 3- ducts                                                     4+ staining                                                                    2-3+                                                                     __________________________________________________________________________

                                      TABLE XIII                                  __________________________________________________________________________    Staining Pattern of 9F7 anti-VEGF by direct immunohistochemical                 staining on 6-8 week old GP tissues                                                              Purified GV97 1 μg/ml                                                                           0.5                                 TISSUE        20 μg/ml                                                                          10 μg/ml                                                                          5 μg/ml                                                                           2 μg/ml                                                                           μg/ml                                                                          9F7 supt.                                                                          3F9 supt.                                                                           5F9 supt.            __________________________________________________________________________    DIGESTIVE SYSTEM                                                                LIVER  2+  1-2+ +/- +/-  1-2+ 1-2+                                            INTESTINE 4+ 3+ 2+ 1+  4 + m 4 + m                                                   lymphoid, lymphoid,                                                           rest diff. rest diff.                                                         than than                                                              PANCREAS 1+ of many                                                            and 3+ in                                                                     islands of                                                                    cells                                                                        SMALL 4+ of many 2-3+ of many 1-2+ of +/- of many   3+ of some 3+ of                                                                 some                   INTESTINE and 4+ in and 4+ in many and 4+ and 4+ in   and 4+ in and 4+                                                               in                      lymphoid lymphoid, in lymphoid,   lymphoid lymphoid                            rest diff. lymphoid, rest diff.                                               than fVIII rest diff. than fVIII                                               than fVIII                                                                 STOMACH 3-4+ 1-2+ on most +/- on most +/- on most   3-4+ (some 3-4+                                                                  (some                    occasional occasional occasional   fVIII-ve) fVIII-ve)                        3+ 2+ 1+                                                                    REPRODUCTIVE SYSTEM                                                           TESTIS                                                                        MUSCLE AND                                                                    INTEGUMENTARY SYSTEM                                                          HEART                                                                        ve                                                                            ve                                                                            ve                                                                            ve   3-4+ (some 3-4+ (some                                                     MUSCLE       fVIII-ve) fVIII-ve)                                              SKIN 1-2+ in 1+ in fatty +/- in +/- in fatty 3+                                fatty layer and 3- fatty layer layer and 1-                                   layer and 4+ in and 3-4+ of 2+ of a few                                       3-4+ in cellular a few in in cellular                                         cellular layer cellular layer                                                 layer  layer                                                                 IMMUNE SYSTEM                                                                 SPLEEN 4+ 3+ 2+                                                              ve   4+ 4+                                                                     THYMUS                                                                        URINARY SYSTEM                                                                KIDNEY glomeruli glomeruli 3- glomeruli glomeruli 1-   glomeruli                                                                     glomeruli                                                                       4+ 4+ 2-3+ 2+                                                                3-4+ 3-4+                                                                     ENDOCRINE                                                                    SYSTEM                 ADRENAL                                                                       RESPIRATORY SYSTEM                                                            LUNG                                                                          NERVOUS SYSTEM                                                                CEREBELLUM 4+ 2+ +/- of most +/- of most   4+ 4+                                 and 1+ of a and 1+ of a                                                       few few                                                                    TUMORS                                                                        TUMOR 4+ 4+ 3-3+ 2-3+(2)  4+  3+                                            __________________________________________________________________________

Lack of Reactivity of GV97 With Soluble Human VEGF

GV97 did not bind to recombinant VEGF-coated ELISA plates, nor didrecombinant human VEGF bind to GV97 (ATCC HB-12451) coated ELISA plates.Soluble recombinant human VEGF did not block the binding of 5 μg/ml GV97to tumor endothelium in histological sections even when added at 50μg/ml.

These data suggest that GV97 (ATCC HB-12451) recognizes an epitope ofVEGF that is concealed in recombinant human VEGF but which becomesaccessible when VEGF binds to its receptor on endothelial cells.

GV97 Localization After Injection in Line 10-Bearing Guinea Pies

In contrast with staining data obtained from histological sections(Table XIV, column 1), GV97% antibody (ATCC HB-12451) localizedselectively to tumor endothelial cells after injection into line 10tumor-bearing guinea pigs (Table XIV, column 2). Staining of endothelialcells in the tumor was moderately strong whereas staining of normalendothelium in miscellaneous organs was undetectable.

Anti-bFGF Antibodies Selectively Bind to Tumor Endothelial Cells

GF67 (ATCC HB-12451) and GF82, which had been raised against guinea pigbFGF N-terminus, bound strongly to endothelial cells in frozen sectionsof guinea pig line 10 tumor and to endothelial cells in two types ofhuman malignant tumors (Table XV). By contrast, relatively weak stainingof endothelial cells in miscellaneous guinea pig normal tissues wasobserved.

                                      TABLE XIV                                   __________________________________________________________________________    GV97 injected into tumor bearing GP                                                                           GV 97 20 μ/ml serum                          TISSUE GV97 10 μg/ml volume injected                                     __________________________________________________________________________    DIGESTIVE SYSTEM                                                                LIVER 2+                                                                     ve                                                                             INTESTINE 3+ possible 0.5-1+ of a                                               few                                                                         PANCREAS +/- of many and 2+ in possible 0.5-1+ of a                            islands of cells few                                                         SMALL INTESTINE 2-3+ of many and 4+ in                                         lymphoid, rest diff.                                                          than fVIII                                                                   STOMACH 1-2+ on most occasional possibly 0.5+ of a few                        REPRODUCTIVE SYSTEM                                                           TESTIS                                                                        MUSCLE AND INTEGUMENTARY SYSTEM                                               HEART                                                                        ve                                                                            ve                                                                             MUSCLE                                                                       ve                                                                             SKIN 1+ in fatty layer and                                                     3-4+ in cellular layer                                                       IMMUNE SYSTEM                                                                 SPLEEN 3+ possibly a few 1+                                                   URINARY SYSTEM                                                                KIDNEY glomeruli 3-4+                                                         ENDOCRINE SYSTEM                                                              ADRENAL 4+                                                                   ve                                                                             RESPIRATORY SYSTEM                                                            LUNG 2+                                                                      ve                                                                             NERVOUS SYSTEM                                                                CEREBELLUM 2+                                                                ve                                                                             TUMORS                                                                        TUMOR 4+ 2-4+                                                               __________________________________________________________________________

                  TABLE XV                                                        ______________________________________                                        Anti-GP FGF Antibody Staining on GP Tissues                                     GP TISSUE                GF 67    GF 82                                     ______________________________________                                        DIGESTIVE SYSTEM                                                                LIVER ND ND                                                                   INTESTINE +/- +/-                                                             PANCREAS 2-3+ 2+                                                              SMALL INTESTINE +/- +/-                                                       STOMACH ND ND                                                                 REPRODUCTIVE SYSTEM                                                           TESTIS ND ND                                                                  MUSCLE AND INTEGUMENTARY SYSTEM                                               HEART 2-3+ 1+                                                                 MUSCLE +/- 1+                                                                 SKIN ND ND                                                                    IMMUNE SYSTEM                                                                 SPLEEN 3+                                                                    ve                                                                             THYMUS                                                                        URINARY SYSTEM                                                                KIDNEY 1-2+                                                                  ve                                                                             ENDOCRINE SYSTEM                                                              ADRENAL 1-2+ +/-                                                              RESPIRATORY SYSTEM                                                            LUNG 1-2+ 2-3+                                                                NERVOUS SYSTEM                                                                CEREBELLUM 1+ -1+                                                             TUMORS                                                                        LINE 1 TUMOR 4+ 4+                                                            HUMAN TUMORS                                                                  PHEOCHROMO CYTOMA 4+ 4+                                                       SCHWANOMA 4+ 4+                                                             ______________________________________                                    

EXAMPLE VI Thrombosis of Line 10 Tumor Vasculature by Administration ofGV97-ricin A-chain Immunotoxin to Guinea Pigs

This example describes the effects observed following the administrationof GV97-ricin A-chain immunotoxin to guinea pigs in vivo.

GV97 (ATCC HB-12451) was conjugated to deglycosylated ricin A-chainusing previously published methods (Thorpe et al., 1988). Guinea pigshaving a 2 cm diameter subcutaneous line 10 tumor were injectedintravenously with 700 μg/kg of this immunotoxin. The tumor and variousnormal tissues were removed 24 hours after injection. Killing ofendothelial cells throughout the tumor plus thrombosis of the tumorvasculature was apparent. No damage to any normal tissues was observed.The effect was specific; no damage to tumor endothelium was observed inrecipients of a class-matched (IgM) immunotoxin of irrelevantspecificity.

It is important to note that no toxicity to the guinea pigs wasobserved. The treated animals maintained normal appearance, activity,and body weight.

REFERENCES

The following references are hereby incorporated herein by reference, tothe extent that they explain, further enable, provide a basis for ordescribe the subject matter to which is referred to in thespecification.

Abrams, P. W. et al. (1985) Monoclonal antibody therapy of human cancer(Foon and Morgan, eds.), Martinus Nijhoff Publishing, Boston, pp.103-120.

Anegon, I. (1988) J. Exp. Med. 167(2):452-472.

Ausubel et al. (1989) Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y.

Bauer, T. et al., (1991) Vox Sang, 61:156-157.

Baxter, L. T. et al. (1991) Micro. Res., 41(1):5-23.

Baxter, L. T. et al. (1991) Micro. Res., 41(1):5-23.

Bellon, T., (In press, 1993) Eur. J. Immunol.

Bevilacqua, M. P., et al. (1987) Proc. Natl. Acad. Sci. USA,84:9238-9242.

Bhattacharya, A., et al. (1981) J. Immunol., 126(6):2488-2495.

Bigler, S. A., et al. (1993) Hum. Pathol. 24:220-226.

Billington, W. D. et al. (1986) J. Reprod. Immunol., 9:155-160.

Bindon, C. I. (1988) Eur. J. Immunol., 18:1507-1514.

Bittner et al., (1987) Methods in Enzymol., 153:516-544.

Bjorndahl, et al. (1989) Eur. J. Immunol., 19:881-887.

Bjorntorp, et al. (1983) J. Lipid Res., 24:105-112.

Blakey, D. C., et al. (1987b) Biochem Biophys ACTA, 923Y(1):59-65.

Blakey, D. C., et al. (1987a) Cancer Res., 47:947-952.

Blanchard, D. K., et al. (1988) Cancer Res., 48:6321-6327.

Borden, E. C., et al. (1990) Cancer, 65:800-814. Boyer, C. M., et al.(1989) Cancer Res., 49:2928-2934.

Boyer, C. M., et al. (1989) Int. J. Cancer, 43:55-60.

Buthring, H. J., et al. (1991) Leukemia, 5:841-847.

Burchell et al., (1983) J. Inmunol., 131(1):508-13.

Burrows, F. J., et al. (1993) Proc. Natl. Acad. Sci., 90:8996-9000.

Burrows, F. J., et al. (1992) Cancer Res., 52:5954-5962.

Burrows, F. J., et al. (1991) Cancer Res., 51:4768-4775.

Byers, V. S., et al. (1989) Cancer Res., 49:6153-6160.

Byers, V. S., et al. (1988) Immunol., 65:329-335.

Capobianchi, M. R. (1985) Hum. Immunol., 13:1.

Cheifetz, S. et al. (1992) J. Biol. Chem., 267:19027-19030.

Chen, T. Y., et al. (1990) J. Immunol., 145:8-12.

Cheresh, D. A., (1987) Proc. Natl. Acad. Sci. USA, 84:6471-6475.

Cherwinski, H. M. (1987) J. Exp. Med., 166:1229-1244.

Cherwinski et al. (1989).

Chodak, G. W., et al. (1980) Ann Surg., 192:762-771.

Colberre-Garapin et al., (1981), J. Mol. Biol., 150:1.

Colcher et al., (1987) Cancer Res., 47:1185; and 4218.

Collins, T. et al. (1984) Proc. Natl. Acad. Sci. U.S.A., 81:4917-4921.

Cotran, R. S., et al. (1986) J. Exp. Med., 164:661-666.

Daar, A. S., et al. (1984) Transplantation, 38(3):293-298.

Darzynkiewicz, Z., et al. (1976) Proc. Natl. Acad. Sci. USA,73:2881-2884.

de Waal, R. M. W. (1983) Nature, 303:426-429.

DeFranco, A. L. (1991) Nature, 352:754-755.

Demur, C. , et al. (1989) Leuk. Res., 13:1047-1054.

Denekamp, J. et al. (1982) Brit. J. Cancer, 461:711-720.

Denekamp, J. (1984) Prog. Appl. Microcirc., 4:28-38.

Denekamp, J. (1986) Cancer Topics, 6:6-8.

Denekamp, J. (1990) Cancer Meta. Rev., 9:267-282.

Denekamp, J. (1990) Cancer Netastasus Rev., 9:253-266.

Dillman, R. O., et al. (1988) Antibody, Immunocon. Radiopharm., 1:65-77.

Dixon et al., Mol. & Cell Biol., 7:4896-4902, 1989.

Duijvestijn, A. M., et al. (1987) J. Immunol., 138:713-719.

Duijvestijn, A. M., et al. (1986) Proc. Natl. Acad. Sci. (USA),83:9114-9118.

Dunham L. C. et al. (1953) Cancer Inst., 13:1299-1377.

Dustin, M. et al. (1986) J. Immunol., 137:245-254.

Dvorak, H. F. et al. (1991) Cancer Cells, 3(3):77-85.

Enestein, J. et al. (1992) Exp. Cell. Res., 203:499-503.

Engert, A., et al. (1991) Leuk. Res., 15:1079-1086.

Epenetos, A. A., et al. (1986) Cancer Res., 46:3183-3191.

Fernandez-Botran, R. et al. (1991) FASEB J., 5:2567-2574.

Fett, J. W. et al. (1987) Biochem. Biophys. Res. Comm., 146:1122-1131.

Fett, J. W., et al. (1985) Biochem., 24:5480-5486.

Flickinger & Trost, (1976) Eu. J. Cancer, 12(2):159-60.

Folkman, J. (1990) J. Natl. Cancer Inst., 82:4-6.

Folkman, J. (1985) Adv. Cancer Res., 43:175-230.

Folkman, J. (1985) In: V. T. DeVita, S. Hellman and S. A. Rosenberg(eds.), Important Advances in Oncology, Part I, pp. 42-62, Philadelphia:JB Lippincott.

Fox, B. A. et al. (1990) J. Biol. Resp., 9:499-511.

French, J. E., et al. (1963) Br. J. Exp. Pathol., XLV:467-474.

Fujihara, K., (1991) Nippon Jibiinkoko Gakkai Kaiho, 94:1304-1314.

Fujimori, K. et al. (1989) Cancer Res., 49:5956-5663.

Fulton, R. J. et al. (1986) J. Biol. Chem., 261:5314-5319.

Garcia, F. U. et al. (1990) Am. J. Pathol., 136:1125-1135.

Garrido, M. A., et al. (1990) Cancer Res., 50:4227-4232.

Gerlach, H., et al. (1989) J. Exp. Med., 170:913-931.

Ghetie, M. A., et al. (1988) Cancer Res., 48:2610-2617.

Ghetie, M. A., et al. (1991) Cancer Res., 51:5876-5880.

Ghose, et al. (1987) CRC Critical Reviews in Therapeutic Drug CarrierSystems, 3:262-359.

Ghose, et al. (1983) Meth. Enzymology, 93:280-333.

Girling, et al. (1989) J. Int. J. Cancer, 43:1072-1076.

Glennie, M. J., et al. (1987) J. Immunol., 139:2367-2375.

Gomez-Pinilla and Cotman, Neuroscience, 49:771-780, 1992.

Gospodarowicz, D. et al. (1981) J. Cell. Physiol., 107(2):171-183.

Gospodarowicz, D. et al. (1979) Exp. Eye Res., 29(5):485-509.

Gougos, A. & Letarte, M. (1990) J. Biol. Chem., 265:8361-8364.

Gougos, A. & Letarte, M. (1988) J. immunol., 141:1925-1933.

Gougos, A. et al. (1992) Int. Immunol., 4:83-92.

Greiner, J. W. et al. (1991) J. Surg. Oncol. (Suppl.), 2:9-13.

Griffin, T. W., et al. (1989) J. Cancer Immunol. Immunother., 29:43-50.

Griffin, T. W., et al. (1988) Treat. Res., 37:433-455.

Groenewegen, G., et al. (1985) Nature, 316:361-363.

Gross, J. A., et al. (1990) J. Immunol., 144:3201-3210.

Hagemeier, H. H., et al. (1986) Int. J. Cancer, 38:481-488.

Hailing et al. (1985) Nucl. Acids Res., 13:8019-8033.

Hammerling, G. J. (1976) Transplant. Rev., 30:64-82.

Harkness, J. E. (1983) The Biology and Medicine of Rabbits, (Lea &Fabinger, Philadelphia).

Herbin and Gross, "Binding sites for bFGF on solid tumors are associatedwith the vasculature," In Angiogenesis: Key Principles, R-Steiner, P.Weiss & R. Langer, eds. Birkamser Verlag, Basel, Switzerland, 1992.

Hess, A. D., et al. (1991) Transplantation, 6:1232-1240.

Hokland, M. et al. (1988) Cancer Meta. Rev., 7:193-207.

Horak, E. R. et al. (1992) Lancet, 340:1122-1124.

Imai, Y., et al. (1991) J. Cell Biol., 113:1213-1221.

Inouye et al., (1985), Nucleic Acids Res., 13:3101-3109

Irie, R. F., et al. (1970) J. Natl. Cancer Inst., 45:515-524.

Irie, R. F. (1971) Cancer Res., 31:1682-1689.

Jaffe, E. A. et al. (1973) J. Clin. Invest., 52:2745-2752.

Jain, R. K. (1990) Cancer Meta. Rev., 9(3):253-266.

Jain, R. K. (1988) Cancer Res., 48:2641-2658.

Jones, E. L. et al. (1984) J. Pathol., 144:131-137.

Jones, P. L., et al. (1986) Cancer Immunol. Immunother., 22:139-143.

June, C. H., et al. (1987) Molecular Cell Biology, 12:4472-4481.

Juweid, M. et al. (1992) Cancer Res., 52:5144-5153.

Kandel, et al. (1991) Cell, 66:1095-1104.

Kaplan, E. H. (1989) Hematology/Oncology Clinics of North America,3:125-135.

Karasek, M. A. (1989) J. Invest. Derm., 93(2, suppl):335-385.

Kasahara, T., (1983) J. Immunol., 131(5):2379-2385.

Kennel, S. J., et al. (1991) Cancer Res., 51:1529-1536.

Kim K. J., (1979) J. Immunol., 122(2):549-554.

Kimura, S., et al. (1980) Immunogenetics, 11:373-381.

Knowles & Thorpe (1987) Anal. Biochem., 120:440-443.

Kobayashi, M. et al. (1991) Human Cell, 4:296-305.

Kohler, G. & Milstein, C. (1975) Nature, 256:495-497.

Koulova, L., et al. (1991) J. Exp. Med., 173:759-762.

Lamb et al. (1985) Eur. Jrnl. Biochem., 148:265-270.

Lan, M. S., et al. (1987) Int. J. Cancer, 39:68-72, 1987.

Lee, W. T., et al. (1990) J. Immunol., 144:3288-3295.

Li, J-l., et al. (1989) Cell. Immunol., 118:85-89.

Logan et al., (1984), Proc. Natl. Acad. Sci. USA, 81:3655-3659.

Lord et al. (1992), In Genetically Engineered Toxins (Ed. A. Frank, M.Dekker Publ., p. 183)

Lowder, J. N. et al. (1987) Blood, 69:199.210.

Lowe, J., Ling, et al. (1986) Immunol Lett., 12:263-269.

Lowy et al., (1980), Cell, 22:817.

Madri, J. A. et al. (1992) Mol. Reprod. Devel., 32:121-126.

Maeda, K. et al. (1991) J. Invest. Derm., 97:183-189.

Mason, D. W. & Williams, A. F. (1980) Biochem. J., 187:1-20.

Matsuzaki et al., Proc. Natl. Acad. Sci. USA, 86:9911-9915, 1989.

Mazzocchi, A. et al. (1990) Cancer Immunol. Immunother., 32:13-21.

Metcalf, D. and Nicola, N. A. (1985) In: Molecular Biology of TumorCells (Wahren, B. et al., Eds) New York: Raven Press, pages 215-232.

Mignatti, P. et al. (1991) J. Cell. Biol., 113:1193-1201.

Miotti et al., (1987) Int. J. Cancer, 39:297.

Morrow, K. J. Jr., et al., (1991) Techniques for the production ofMonoclonal and Polyclonal Antibodies. In Colloidal Gold:

Principles, Methods and Applications, Vol. 3, Ed. Hayatt, pp. 31-57,Academic Press, San Diego, Calif.

Mulligan et al., (1981), Proc. Natl. Acad. Sci. USA, 78:2072.

Murray, J. C., et al. (1989) Radio. Onc., 16:221-234.

Murray et al. (1984) N. Eng. Jrnl. Med., 310:883.

Naieum, M. et al. (1982) J. Immunol. Methods, 50:145-160.

Natali, P. G., et al. (1981) Scand. J Immunol., 13:541-546.

Nawroth, P., et al. (1988) J. Exp. Med., 168:637-648.

Nevens, J. R. (1992) J. Chromatography, 597:247-256.

Nishikawa et al., Advances in Experimental Medicine and Biology,324:131-139, 1992.

Novick, D. et al. (1989) J. Exp. Med., 170:1409-1414.

O'Connell, K. A. et al. (1990) J. Immunol., 144(2):521-525.

O'Connell, P. J. et al. (1992) Clin. Exp. Immunol., 90:154-159.

Ogata et al. (1990) J. Biol. Chem., 256:20678-20685).

O'Hare et al. (1987) FEBS Lett., 210:731.

O'Hare et al., (1981), Proc. Natl. Acad. Sci. USA, 78:1527.

Oi & Morrison (1986) Mt. Sinai J. Med., 53(3):175-180.

Oi, V. T. et al. (1978) Curr. Top. Microbiol. Immunol., 81:115-120.

Osborn, L. et al. (1989) Cell, 59:1203-1211.

Palleroni, A. V., et al. (1991) Int. J. Cancer, 49:296-302.

Perez, P., et al. (1985) Nature, 316:354-356.

Peri, G. et al. (1990) J. Immunol., 144(4):1444-1448.

Pervez, S., et al. (1988) Int. J. Cancer, 3:30-33.

Pietersz, G. A., et al. (1988) Antibody, Immunoconj. Radiopharm.,1:79-103.

Pober, et al. (1983) J. Exp. Med., 157:1339-1353.

Pober, J. S., et al. (1991) J. Immunol., 137:1893-1896.

Pober, J. S., et al. (1987) J. Immunol., 138:3319-3324.

Qian, J. H., et al. (1991) Cancer Res., 140:3250.

Reilly et al., Biochem. Biophys. Res. Commun., 164:736-743, 1989.

Reisfeld et al., (1982) Melanoma Antigens and Antibodies, p. 317.

Rettig, W. J. et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10832-10836.

Revtyak, G. E. et al. (1988) American J. of Physiology, 254:C8-19.

Rice, G. E. et al. (1990) J. Exp. Med., 171:1369-1374.

Rowlinson-Busza, G. et al. (1991) Cancer Res., 51:3251-3256.

Ruco, L. P., et al. (1990) Am. J Pathol., 137(5):1163-1171.

Ruther et al., (1983), EMBO J., 2:1791

Rybak, S. M. et al. (1987) Biochem. Biophys. Res. Comm., 146:1240-1248.

Sambrook et al., (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, N. Y.

Sanchez-Madrid, F. (1983) J. Immunol., 130(1):309-312.

Sands, H. (1988) "Immunoconjugates and Radio-pharmaceuticals",1:213-226.

Sands, H. (1988) Antibody, Immunoconjugates and Radio-pharmaceuticals,1:213-226.

Santerre et al. (1984) Gene, 30:147.

Sarma, V. et al. (1992) J. Immunology, 148:3302-3312.

Schlingemann, R. O., et al. (1985) Lab. Invest., 52:71-76.

Schutt, C. , et al. (1988) Immunol. Lett., 19:321-328.

Seto, M., et al. (1982) J. Immunol., 128:201-205.

Shen, G. L., et al. (1988) Int. J. Cancer, 42:792-797.

Shepard et al., (1991) J. Clin. Immunol., 11:117-127.

Shockley, T. R. et al. (1991) Ann. N.Y. Acad. Sci., 617:367-382.

Sillman, F. et al. (1981) Am. J. Obstet. Gynecol., 139:154-159.

Smith et al., (1983), J. Virol., 46:584.

Spertini, O., et al. (1991) J. Immunol., 147:2565-2573.

Spitler, L. E. (1988) Cancer Treat. Res., 37:493-514.

Steel, G. G. (1977) Growth Kinetics of Tumours, p.6, Clarendon Press,Oxford, U.K.

Stevenson, F. K. et al. (1990) Chem. Immunol., 48:126-166.

Stoscheck, C. M. and King, L. E. (1986) Cancer Res., 46:1030-1037.

Stout, R. D. & Suttles, J. T. (1992) Cell. Immunol., 141:433-443.

Street, N. et al. (1989) Cell. Immunol., 120:75-81.

Sung, C. et al. (1990) Cancer Res., 7382-7392.

Szybalska et al., (1962), Proc. Natl. Acad. Sci. USA, 48:2026.

Takac, L., et al. (1988) J. Immunol., 141:3081-3095.

Tax, W. J. et al. (1984) Clin. Exp. Immunol., 55:427-436.

Tazzari, P. L. et al. (1987) Clin. & Exp. Immunol., 70:192-200.

Thompson, C. B., et al. (1989) Proc. Natl. Acad. Sci. USA, 86:1333-1337.

Thor et al., (1987) Cancer Res., 46:3118.

Thorpe et al., (1988) Cancer Res., 48:6396-6403.

Thorpe, P. E., et al. (1985) Eur. J. Biochem., 147:197-206.

Thorpe, P. E., et al. (1988) Cancer Res., 48:6396-6403.

Thorpe, P. E., et al. (1987) Cancer Res., 47:5924-5931.

Thorpe, P. E., et al. Selective killing of proliferating vascularendothelial cells by an anti-fibronectin receptor immunotoxin, Presentedat the 16th LH Gray Conference, University of Manchester Institute ofScience and Technology, Sep. 17-21, 1990.

Till, M., et al. (1988) Cancer Res., 48:1119-1123.

Titus, J. A. et al. (1987) J. Immunol., 138:4018-4022.

Tumor Blood Circulation (1978) CRC Press Inc., Boca Raton.

Tutt, A. et al. (1991) Eur. J. Immunol., 21:1351-1358.

Vaickus, L., et al. (1991) Cancer Invest., 9:195-209.

Van Deurs, B., et al. (1988) J. Cell Biol., 106:253-267.

Van Deurs, B., et al. (1986) J. Cell Biol., 102:37-47.

Van Duk, J. et al. (1989) Int. J. Cancer, 43:344-349.

Van Heeke et al., (1989), J. Biol. Chem., 264:5503-5509.

Vitetta, E. S. et al. (1991) Cancer Res., 15:4052-4058.

Vogel & Muller-Eberhard (1981) Anal. Biochem., 118(2):262-268.

Wang, J. M. et al. (1993) Int. J. Cancer, 54:363-370.

Watanabe, Y. et al. (1988) Eur. J. Immunol., 18:1627-1630.

Watanabe, Y., et al.(1989) Proc. Natl. Acad. Sci. USA, 86:9456-9460.

Weidner, N. et al. (1992) J. Natl. Cancer Inst., 84:1875-1887.

Weiner, L. M., et al. (1989) Cancer Res., 49:4062-4067.

Westphal, J. R. et al. (1993) J. Invest. Derm., 100:27-34.

Wigler et al., (1980), Proc. Natl. Acad. Sci. USA, 77:3567.

Wigler et al., (1977), Cell, 11:223.

Winter, et al. (1991) Nature, 349:293-299.

Wu, M., Tang, et al. (1990) Int. J. Pharm., 12:235-239.

Xu et al., J. Biol. Chem., 267:17792-17803, 1992.

Yamaue, H. et al. (1990) Biotherapy, 2:247-259.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Ala Pro Met Ala Glu Gly Glu Gln Lys Pro Ar - #g Glu Val Val Lys        Phe                                                                             1               5   - #                10  - #                15              - - Met Asp Val Tyr Lys Arg Ser Tyr Cys                                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Ala Pro Met Ala Glu Gly Gly Gly Gln Asn Hi - #s His Glu Val Val Lys      1               5   - #                10  - #                15               - - Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys                                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Al - #a Leu Pro Glu Gly Gly      1               5   - #                10  - #                15               - - Asp Gly Gly Ala Phe Ala Pro Gly Cys                                                  20      - #            25                                        __________________________________________________________________________

What is claimed is:
 1. A method for treating a vascularized tumor,comprising administering to an animal having a vascularized tumor abiologically effective amount of a conjugate comprising a selectedtherapeutic agent linked to an antibody that binds to a complex of agrowth factor and a growth factor receptor present on the surface ofintratumoral blood vessels of the vascularized tumor, but that does notbind to the individual growth factor or growth factor receptor.
 2. Themethod of claim 1, wherein the conjugate comprises a monoclonal antibodyor monoclonal antibody fragment.
 3. The method of claim 1, wherein theantibody binds to the same epitope as the monoclonal antibody GV39 (ATCCHB-12450).
 4. The method of claim 1, wherein the antibody binds to thesame epitope as the monoclonal antibody GV97 (ATCC HB-12451).
 5. Themethod of claim 1, wherein the antibody binds to a complex of FGF and anFGF receptor.
 6. The method of claim 1 wherein the antibody binds to acomplex of TGFβ and a TGFβ receptor.
 7. The method of claim 1, whereinthe therapeutic agent is an anticellular agent capable of killing orsuppressing the growth or cell division of tumor-associated endothelialcells of intratumoral blood vessels.
 8. The method of claim 7, whereinthe anticellular agent is a chemotherapeutic agent, a radioisotope, or acytotoxin.
 9. The method of claim 8, wherein the anticellular agent is asteroid, a cytokine, an antimetabolite, an anthracycline, a vincaalkaloid, an antibiotic, an alkylating agent, or an epipodophyllotoxin.10. The method of claim 8, wherein the anticellular agent comprises aplant-, fungus- or bacteria-derived toxin.
 11. The method of claim 10wherein the toxin comprises an A chain toxin, a ribosome inactivatingprotein, α-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheriatoxins Pseudomonas exotoxin, bacterial endotoxin or the lipid A moietyof bacterial endotoxin.
 12. The method of claim 11 wherein the toxincomprises deglycosylated ricin A chain.
 13. The method of claim 1,wherein the antibody binds to a complex of a growth factor and a growthfactor receptor, the presence of which on the intratumoral blood vesselsis cytokine-inducible.
 14. The method of claim 1, further comprisingadministering an antitumor antibody-therapeutic agent conjugate to theanimal.
 15. The method of claim 14 wherein the antitumorantibody-therapeutic agent conjugate comprises an HMFG-2, SM-3, B72.3,PR5C5, PR4D2, 9.2.27, OV-TL3, MOv18 or anti-p185^(HER2) antitumorantibody or fragment thereof.
 16. The method of claim 1, wherein theanimal is a human cancer patient.